472B Lab Manual 2ndEd

7/30/2019 472B Lab Manual 2ndEd

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Chemistry 472BBIOTECHNOLOGY

LABORATORY

MANUAL

Mark Brandt , Ph.D.Second editionJ a nuar y , 2002


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Table of Contents

Int roduction to th e Labora tory.................................................................................... 1G enera l Informat ion: Keeping a la bora tory notebook... . .. .. . .. .. . .. .. . .. .. . .. .. . .. .. . .. .. . .. .. .. 3G enera l Information: La bora tory reports.. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 5

B a sic La bora tory Techniq ues: P ipett ing................................................................... 14B a sic La bora tory Techniques: Measuring a bsorba nce.... . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. 16B a sic La bora tory Techniq ues: P erforming dilutions............................................... 18B a sic La bora tory Techniq ues: B uffers....................................................................... 20G enera l In forma tion: Molecular biology...................................................................... 22G enera l In forma tion: Cell genotypes........................................................................... 24Methods: P olymera se cha in reaction.......................................................................... 26Methods: P la smid prepara tion...................................................................................... 29

P rocedure for P la smid miniprep:...................................................................... 31Methods: Liga tion............................................................................................................ 32Methods: Competent cell prepa ra tion and Tra nsforma tion... .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. 34Methods: Selection an d Screening................................................................................ 35

G eneral In forma tion: R estriction digestion................................................................ 38Electrophoretic Techniq ues: SD S P AG E.................................................................... 39Electrophoretic Techn iques: Aga rose gel electrophoresis....................................... 44Electrophoretic Techniq ues: DNA sequencing.......................................................... 46Electrophoretic Techniq ues: Western blott ing.......................................................... 49

P rocedure for running t he SDS P AG E an d blotting:... .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. 50Western blott ing incuba tions............................................................................ 52

Methods: P rotein a ssa ys................................................................................................ 56Methods: P rotein purificat ion........................................................................................ 58

P rocedure for pouring a column........................................................................ 63Methods: Cell lysis for protein purificat ion................................................................. 65Methods: ADP -glucose pyrophosphoryla se a ssa y.................................................... 66

ADP -glucose pyrophosphorylase a ssa y procedure... .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. . .. 68Methods: P rotein cryst a llogra phy................................................................................ 70In tr oduction to enzym e kinetics................................................................................... 74Definitions ......................................................................................................................... 83

Other useful Information:B iochemistr y St ockroom: MH -277Ch emistr y & B iochemistry Office: MH -580


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Introduction to the Laboratory

This course is int ended t o int roduce you to some of th e most w idely used experiment a lprocedures in biotechnology, including DNA isolation, manipulation, cloning, andmut a genesis, and protein purifica tion an d cha ra cteriza tion. You w ill a lso ga in somefam ilia rity w ith some of the t ypes of equipment frequently used in biochemistry a ndmolecular biology.

Resea rch is often a collaborat ive effort in w hich m a ny people ma y cont ribute todifferent a spects of a given project. Few papers in t he scientific litera tur e are w ritt enby s ing le authors ; the vas t ma jor i ty o f pa pers ha ve a t leas t t wo aut hors , and m an ypapers ha ve more tha n ten cont ributin g people. In par t t o provide a more a uth enticexperience of actual lab work, experiments will be done in groups of two or three. Youma y choose par tn ers, or you ca n a sk to be a ssigned to a group.

The biotechn ology la bora tory course, like a ll labora tory courses, is an explora tion of

procedures. This m ean s th a t, in order t o get full benefit from t he course, you w ill needto read the manual, and you should participate as much as possible in thediscussions. You should ask questions in or out of class. You should also try topart icipat e in the actua l la b work (a nd not simply allow y our lab par tn ers to do th ingsfor you). The more effort y ou put int o the cours e work, t he more you w ill lear n. Theclass is a n opportun ity t o lea rn va luable skills; take full a dva nta ge of it!

Prior to many of the lab periods, you will need to spend some time reading theLaboratory Manual. This reading will provide background information and an outlineof the procedures t o be performed. If you do not do this, you w ill find y ourself w a stin glarge amounts of class t ime, and annoying both your lab partners and yourinstr uctor. To encoura ge your understa nding of th e ma teria l, you w ill have problem

sets th at cover ma terial related t o the planned experiments.

The biotechnology laboratory is conducted as a directed research project. Thismean s tha t a lthough th e genera l procedures are well esta blished, th e overall goa l ofea ch experim ent is th e a cquisition of new information. Because of the nature ofscient ific resear ch, predicting t he out come of experiments th a t h a ve not previouslybeen performed is difficult. I t ma y th erefore be necessa ry t o design new experimentsbased on the results of previous ones, or to repeat experiments that yieldeduninterpreta ble or a mbiguous results. If y ou expect t o know w ha t exa ctly y ou w ill bedoing w eeks in a dva nce, you will be in for a shock. On th e oth er ha nd, if you approachth e course w ill an open a nd flexible mindset, you w ill learn how r esea rch is performedin a biotechnology laborat ory.

SAFETY: La bora tories conta in ha za rds of va rious kinds. Everyoneis r equ i r edt owear closed-toe shoes, long pants, goggles with side shields, and a lab coatw hile performing laborat ory w ork. Stud ents should not w ork in the labora tory if theinstr uctor is not present.


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Some of th e chemicals used a re toxic, mut a genic, or t era togenic. If you believe th a tyou ha ve a h ealt h condit ion t ha t put s you at exceptiona l risk, or believe yourself to bepregna nt, plea se see your instructor in privat e to discuss t he issue. If you ha vequest ions or concerns a bout exposure to ha za rdous chemica ls , plea se consult yourinst ructor or go to th e Resea rch a nd In str uctiona l Sa fety Office (MH-557).

PHILOSOPHICAL ISSUES: Scientific research involves an exploration of theunknown . In some classes, a q uestion ha s a sin gle correct a nsw er, which is know nto th e instr uctor , and impa rt ed to th e stud ents. In research, however, the correctanswer is rarely known ahead of t ime, and must instead be inferred from theexperimenta l results . Resear chers must th erefore become accust omed t o some levelof uncer ta inty a bout t he correct a nsw er to an y exper imenta l quest ion, a nd m usta lwa ys rema in open to experimenta l evidence th a t cont ra dicts a hypothesis th a t ha sa risen from previous experiment s. Your t a sk a s a scientist w ill be to consider yourda ta , and to a t tempt to interpret i t . In this context , wrong a nsw ers a re answ erstha t a re contra dicted by your da ta or tha t do not a r ise log ica l ly from the da t a youhave collected.

This uncer ta inty a s to the correct a nsw er means t ha t you must be ca re fu l w henreport ing w ha t you did an d wh a t you observed, especia lly if you observe somethingunexpected. Humans are good at fooling themselves; you need to guard againstreport ing w ha t you expect to seer a t her tha n wha t you ac tua l ly d i dsee. Scientificfraud, in which people intentionally report false data, is considered very seriousbeca use it results in a difficult-to-overcome belief in an a nsw er th a t conflicts w ith th etruth. You will occasionally see retractions, in which a scientist publishes astatement that information in a previously published paper is the result of ana rt ifact , a nd is not a reflection of the correct an sw er. Avoiding th e emba rra ssmentof publishing a retra ct ion is one reason for t he care t ha t people ta ke in performing

experiments a nd in interpreting th e results.

Another ethical issue is th e proper cita tion of th e sources of informa tion you use forany scientific writing. You should always properly reference the authors of papers orbooks you consult . I t a lso means tha t you should cite th e inventors of methods t ha tyou use for your experiments. I f you do not, in effect y ou ar e cla iming credit for w orkperformed by others.


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General Information: Keeping a Laboratory Notebook

All student s w ill be required to ma inta in a labora tory n otebook. The notebook will beused for the recording of laboratory data and calculations, and will be criticallyimporta nt for w riting your lab reports.

The purpose of a laboratory notebook is to allow anyone with some biochemicalknowledge to understand exactly w ha t y ou did. You need to record t he informat ion insuf f icient de ta i l so a s to be a ble to repea t i t , an d you must be able to understa ndexa ctly wh a t y our results w ere. You w ill need good notes to be able to write your la breports ; in a ddition, a s your un dersta nding of biochemistry improves, your n otebookshould a l low you to f igure out wh y some pa rts of your experiments did n ot w ork a sexpected.

Compa nies th a t perform resea rch requir e their employees to keep proper notebooks.In many companies, company policy dictates that any work not recorded in thenotebook w a s, by definition, never a ctua lly performed. As a result , th e work must be

repea ted, w hich tends t o ha ve deleterious effects on th e ca reer opport unit ies of theemployees involved. In ca ses of disputes a s t o priority, notebook da tes a re sometimesused to indica te exact ly wh en a n experiment wa s performed. Ownership of pa tents(and in some cases large amounts of money) can therefore be critically dependent onkeeping a proper n otebook. In str uction in keeping la bora tory notebooks is th erefore ama jor pa rt of most la bora tory courses.

In your notebook, ea ch experiment sh ould begin w ith a title, a date, and a s t a t ementof th e objectiveof th e plan ned w ork. You should a lso record exactly what you didat each step (being sure to mention any th ing tha t you did tha t dif fered from theinforma tion in th e Manua l). In a ddition, you should record a ny n umerica l informa tion,such as the weights of reagents used, absorbance readings, enzyme activities, protein

concentrations, and buffer concentrations.

Most experiments will extend over several days, and over several pages in yournotebook. To allow y ou to keep tr a ck of w ha t you ha ve done, you sh ould include t heda y s da te a t the t op of each page . Inc luding sub-t i t les for ea ch pa ge may ma ke i teasier to keep tr a ck of wh a t you did at each step.

Everything you do should be recorded directly int o your la b notebook in pen. If youma ke a m ista ke, dra w a line th rough it , an d wr ite the correction next to the mistake.(It may turn out that the original information was correct after all, so do notoblitera te th e origina l informa tion by erasin g it , or by removing the page from yournotebook.) Any calculat ions performed should be w ritt en directly int o your book. Any

w ork done on a computer, or printouts from la bora tory inst ruments, sh ould be ta peddirectly int o your la b notebook.

Writing importa nt informa tion on scra p paper, and th en recording it in y our n otebooklater is not acceptable. If you are writing something while in the laboratory,you should be writing it directly into the notebook.


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At ea ch st ep in your experiment (af ter ea ch assa y or measurement ), in ad dit ion tothe results , record your th oughts regarding t he experiment a nd h ow you think i t isgoing. Record your mistakes, and your attempts to rectify them. Record theca lculat ions involved in a ny t ype of dat a a na lysis , a s well a s expla na t ions for bothw ha t you did an d w ha t you think i t mea ns. A resea rch project is a journey into theunknown ; your labora tory notebook is usua lly your only guide th rough th e forests ofuncerta inty.

It is a lso a good idea to look over your notebook periodically dur ing t he semester, a ndma ke notes of th ings th a t you do not understand, so th at you can ask quest ionsbefore the lab r eport s a re due.

Do not sa y w ell , I wil l remember w ha t t his means ; instea d, w r i t e i t d ow n! D o notsay I w ill remember wha t I wa s thinking while I did this experiment; instea d, w r i t e i t d own! I f y ou use your lab n otebook properly, you w ill find t ha t w rit ing y our la breport s is much easier, a nd y ou will be developing good ha bits for the fut ure.


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General Information: Laboratory Reports

The la bora tory reports are ma jor w rit t en assignment s, due a t int ervals during th esemester. The la borat ory report s should be wr itt en in t he form of a scient ific paper.To help you lea rn to w rite a scientific pa per correctly, th e labora tory r eports w ill bedue in t w o sections, w ith th e second report building on t he first one. The second reportshould conta in all of th e informa tion from the previous report , plus all of the neww ork. You should incorporat e th e instr uctors suggestions, using th ese comment s t oguide you in th e genera tion of t he n ew sections. Note tha t the second laborat oryreport w il l be gra ded more str ingent ly t ha n t he f irst one: you a re expected t o learnfrom your mist a kes!

All of the laboratory reports are expected to be well-formatted, word-processeddocuments, written in standard scientific American English. The use of spell-checkersa nd gr a mma r-checkers is str ongly recommen ded. (Note: the Appendix does not ha veto be neat ly as nea t ly forma tt ed as th e rest of th e report , a nd, i f necessary, ma y behandwritten.)

In scientific resear ch, results a re report ed to th e world in th e form of scient ific paperspublished in the peer-reviewed scientific literature. These papers are not onlyimporta nt in disseminat ing th e results of th e resea rch, but a re crit ica l for essentiallya ll a spects of ca reer a dva ncement for the scient ists involved. Learning to write aproper scientific paper is therefore an important part of the education of allscientists.

S cient ific pa pers a re expected t o be wr itt en in a w ell-defined forma t. The overa llformat is generally similar in all journals, although the specific details varysomewha t. In this class, the laborat ory reports should be in t he form of a paper in t heJournal of B iological Chem istry. Looking for papers in the J our nal of B iological

Chemistryto use a s exa mples is strongly recommended. (Note th a t t he forma tt ingtha t you should a t t empt t o emula t e applies to content; you do not need to spendtime genera ting th e specific page la yout of a J our nal of Biological Chemi str ypaper.The preferred pa ge layout for la b report submission ha s t he body of your pa per indouble-spaced text.) In keeping with this formatting, the report should have thefollowing sections: Title Page, Abstract, Introduction, Materials and Methods,Results, D iscussion, a nd Appendix.

Ma ny scient ists ha ve th eir ow n preferred w a ys of writ in g pa pers. Most scient ists ,however, use an it erat ive process of wr iting, in wh ich t hey w rite the paper, and th enrewr ite i t severa l t imes before submit t in g the paper to th e journal for review an d(hopefully) publica tion. In a ddition, most pa pers a re writ ten in a n order tha t devia tes

from the f ina l forma t . A common procedure is to writ e th e Methods sect ion f irst ,followed by the Results section. The Methods section is a simple description ofprocedures a nd ca n be w rit ten before the experimenta l results ha ve been a na lyzed.The Results section contains the observations that constitute the study to bepublished. Once these sections a re w ritt en, most people write a n in complete dr a ft ofth e Discussion section t ha t expla ins th e results in t he context of the pa per.


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After the Results section is written, and some thought put into interpreting theresults, most people write the Introduction. When writing your Introduction, youshould th ink of th e In tr oduction a s a n episode of J eopardy : the Results a re t hea nsw ers, an d now it is necessa ry t o come up wit h corresponding q uestions. You do notneed to write th e quest ions in t he form of a q uest ion, but y ou should think a boutra ising quest ions in th e rea ders mind th at you will then a nsw er in the Results a nd

Discussion sections.

After writing the Introduction, you should then look at how you have writtenInt roduct ion, and rewrit e the Results sect ion to more clea rly a nsw er the quest ionsra ised in th e Intr oduction, an d then w rite the Discussion to interpret a nd clarify thea nsw ers. When properly done, each rewr ite acts a s a n impetus for th e rew rite of adifferent section, until a ll of th e sections fit t ogether int o a coherent st ory.

Fina lly , a f ter a l l of the other sect ions ha ve been wr it ten, you can wr ite the a bstra ct ,by extra ct ing t he most importa nt informa tion from each sect ion an d combining th einforma tion into a single para gra ph.

You sh ould keep these genera l concepts for w riting a paper in m ind w hile consideringthe content of each section. The content of each section of a scientific paper isdiscussed below . (Remember tha t y ou ma y be well advised not t o w rite th e pa per inth is order.)

Title Page: This sh ould include the t it le of your r eport , th e au th ors na me (i .e. yourname), your lab partners name(s), and your address (your e-mail address issufficient).

Abstract: This should be a briefversion of the entire paper. It therefore shouldinclude a br ief int roduction, met hods, resu lts , and d iscussion, expressed in ~ 200

words. This truncation is normally achieved in part by greatly abbreviating themethods portion, unless the methods involved are novel or are crucial tounderstanding the findings presented.

Thousands of papers are published every week. Most literature database searchengines include the tit le and a bstra ct , but do not include the remainder of the paper.In w riting the a bstra ct , remember tha t t he vast m a jority of rea ders probably w ill notread the paper, because they lack the time. Therefore, in order to present yourinforma t ion to the la rgest possible a udience , you need to have a n a bst ra ct t ha t isclear ly wr i t ten, tha t is unders ta nda ble wi th out ha ving to read the paper , and t ha tcont a ins a ll of th e relevant findings from t he paper.

The a bstra ct must include the overall conclusions from the paper; once a ga in, thisis important because you want people to know what you have discovered. Yourjob/gra nt fun din g/promot ions/fa me a nd f ortun e/a bilit y t o do more experimen t s/a bilit yto retir e to the exotic locale of your choice ma y depend on ha ving people und erst a ndw ha t y ou ha ve done. (This a pplies to th e entire paper, but t he a bstra ct t ends to be atlea st skimm ed by va st n umbers of people w ho will never rea d t he paper.)


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Introduction: This section should include background information setting up thescientific problem you are attempting to address and the overall goal of theexperiment s you performed. Wha t is th e hypoth esis you a re testin g? Wha t dir ectlyrelevant information is necessary to understand this hypothesis and why is itimporta nt? Wha t is not known t ha t you hope to a ddress? Wha t a re you pla nning t oa tt empt t o accomplish? (Very br iefly) How did y ou a ccomplish t his?

In w riting a n introduction, you are at tempting to orient t he rea ders, so tha t th ey willknow wh a t t o consider as they rea d t he rest of the paper. This means tha t y ou shouldcarefully consider whether you are presenting information that is irrelevant ormislea ding. If you discuss an issue rela ted to your protein in the introduction, th ereader will expect you to address that issue in the remainder of your paper. Ina ddition, a fter ha ving read y our intr oduction, th e reader should ha ve an a ppreciat ionof th e qu estions you w ere a tt empting to a ddress w ith your experiment s and w hythese questions are important. If someone can read your introduction withoutwa nting t o rea d th e rest of your paper to find the a nsw er to the provocat ive questionstha t y ou ra ised, you have not w ritt en your introduction properly!

Methods: This should be a concisesumma ry of wha t you did. It sh ould include enoughdetail so tha t a ny reasonably int elligent biochemist could repea t your w ork, but n ot aminute-by-minute recitation of the hours you spent performing the experiment. Onecommon mist a ke is to include informa tion th a t belongs in t he Results sect ion; theMeth ods section is for methods. For exa mple, a description of a protein assa y sh oulddescribe the procedure used, but generally should not include a l ist of the sa mplesmeasured in th e assa y . On th e other ha nd, a common mista ke is to fa i l to includesome methods, such a s the techniques used to a na lyze the da ta obta ined during th estudy.

When most people rea d a paper, th ey tend t o skipth e Meth ods sect ion unless th eyneed to know exa ct ly how a n experiment w a s performed. This means th at they w illnot read the Methods unless they do not believe your description in the Resultssection, or because they work in the field and want to see if you used a noveltechnique. Because many people skip the Methods section, the Methods sectionshould only be a description of t he met hods used. With th e possible exception of one-time events such a s plasm id const ructions, it is ra rely a good idea to include results inth e Methods section. If you do include results in t he Methods section, th ese resultsshould be a t least summa rized in the Results section a lso.

The Methods sect ion should a lso conta in t he source of th e importa nt reagent s a ndident ifying informat ion for a ny equipment used. B eca use resea rch reagents of high

qua lity a re ava ilable from ma ny vendors, th e precise source of most rea gents is muchless importa nt th a n i t once w a s. I t is common pract ice, however, t o st a te in t heMethods s ection t ha t , for example, th e ADP -glucose pyrophosphoryla se expressionvector w a s a generous gift of Dr. C . Meyer.

Results: This section sh ould be a d escription of wha t y ou did in words, illustra tedwith f igures and ta bles. I t is not enough m erely t o have severa l figures; you need to


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explain what each figure means. Try to avoid merely listing results in the text;instead, expla in t he findings a nd briefly fit t hem int o the overa ll context of th e paper.

For each set of experiment s, you need to consider t he follow ing q uestions: Wha t a reyou doing? Why a nd h ow a re you doing it? Wha t w a s th e rat iona le for t he methods youemployed? Wha t is t he point of th e experiment you a re a bout to describe? Wha tstra tegy a re you using to address th e experimenta l question you are asking?

None of your a nsw ers to the a bove quest ions sh ould be length y, but you do need toconsider these questions in w riting your report . I t ma y be t ota lly obvious t o you w hyyou performed your brilliant experiment, bu t unless you explain th e purpose an dra tionale behind th e experiment, your flaw less reasoning ma y n ot be obvious t o yourreaders.

Remember t ha t you are t elling a story t o people wh o have not done the experiment s.You cannot assume that the reader will know what you are doing and why. Ina ddit ion, you a re tell ing a story t ha t people w il l be predisposed to disbelieve. You

th erefore need to present your informa tion a s clea rly a s possible. If you do so, peoplewill (a t w orst) understa nd w ha t t hey ar e cr i t icizing, an d (a t best) see tha t you ha veput enough thought and effort into your work as to make it likely that you aretrustworthy.

Wha t da ta do you need to report ? Do not report da ta merely beca use i t is ava ilable.In stea d, report da ta to ma ke a point . You are trying to tell a factual story. Thism ea n s t h a t y ou cannot l ie to your rea ders. On the oth er ha nd, i f you perform anirrelevan t experiment , reporting t he results ma y be confusing . For exam ple, if youperform five SD S-P AG E electrophoresis experiments t ha t sh ow essentia lly the sa meresults, you do not need t o include the r esults of each individua l gel.

In reporting the results of an experiment that yielded numerical data, it is poorwr iting technique to simply list in th e text t he sam e values listed in a t a ble or shownin a gr a ph. The raw numbers a re meaningless unless put int o cont ext. In oth er words,cite in the text only the impor tantnumbers, and explain whythese values areimportant .

For reportin g numbers in the t ext, convert th e numbers to reasona ble values. Anu mb er s uch a s 0.0014567 mg/l is not reasonable for two reasons: 1) converting theva lue to 1.4567 mg/ml results in a num ber th a t is m uch easier to rea d, a nd 2) th enum ber of significa nt figures reported seems excessive (unless y ou really believeth a t your experiment w a s a ccura te to five significa nt figures).

As a n exa mple, you w ill be writ ing a description of ADP -glucose pyrophosphorylasepurification and enzyme assays in your Results section. You should consider thefollow ing in wr iting t his section.

Restriction analysis: wh y did you digest t he DNA w ith t hese enzymes? Wha t ba ndsizes did you obta in? Wha t ba nd sizes did you expect t o obta in? Wha t does this t ellyou about w hether t he plasmid is th e correct one?


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Purification: Why did you perform th e purificat ion? Wha t str a tegy d id you employfor t he purifica tion? Why d id you use the steps you used an d not others? Dur ing th epurifica tion, wh a t step resulted in t he great est purifica tion? When did you observe theADP-glucose pyrophosphorylase to elute from the column? Was this expected,unexpected, or did you ha ve no ba sis for ma king a prediction? Is th ere a figure youcould genera te t o clar ify your results? (Is a figure necessar y t o clar ify your results?)

B a sed on your da ta , wa s your purifica tion successful or un successful? Why? D o youha ve a ny da ta other tha n fold-purification to indica te wh ether your purification wa ssuccessful? How did your purification compare to literature values obtained forsimilar proteins?

One import a nt fea tur e in scientific papers describing protein purificat ion is a t a bleth a t present s da ta concerning t he success of the purif ica t ion procedure. This t a bleusually has two goals: 1) to measure the removal of contaminants during thepurificat ion, an d 2) to a ssess t he efficiency of each st ep, a nd t he overa ll efficiency ofthe entire purification process.

The typical purification table for an enzyme gives the values listed for each stepduring t he purification. In order t o set up a purifica tion ta ble, you will need to knowth e enzy me a ctivit y (corr ected to nmol/min of product formed per l of enz ym esolution). For the ADP-glucose pyrophosphorylase assay, the activity can beca lculat ed by:

In the equat ion a bove, sta nda rd C P M/nmol is t he CP M mea sured for t hera dioa ctive sta nda rds on the da y of th e experiment . Although the tota l assa y volume

is 1.0 ml, you will meas ure th e rad ioa ctivity in only 0.5 ml. You w ill usua lly be using10 l of th e enzym e; to correct t o per l of enzym e, you th erefore mu st mult iply by0.1. You w ill usua lly be running the a ss a y for 10 m inutes, a nd t herefore need t omultiply by 0.1 to find the activity per minute. If you diluted your enzymeprepa ra tion, you will need to mult iply by th e dilution fa ctor. You w ill be performingthis calculation repeatedly during the course; you should make certain that youundersta nd th e purpose of ea ch term with in the equa tion.

Total Enzyme Activity: th e activity per l of enzym e solution multiplied by th e tota lvolume of th a t fra ction. (If you ha ve 50 ml of the original homogenat e, the volume is50 ml, even if you only sa ved 0.5 ml for t he a ssa y.)

Total Protein: th e protein concent ra t ion per m l of solut ion m ult iplied by t he tota lvolume of that fraction.

Specific activity: Tota l Activit y/Tota l P rotein (or [Act ivi ty/l]/[P rot einconcent ra t ion in g/l])

Fold-purification: (Specific a ctivit y at a g iven step)/(S pecific a ctivity of sta rtingsample)


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%Yield: (Tota l act ivit y a t a given st ep)/(Tota l act ivit y of sta rt ing sa mple)*100 (Note:th is is int ended to t ra ck t he a mount of ADP -glucose pyrophosphoryla se present inyour sample, a nd not the t otal a mount of all of the proteins in t he fra ction.

Which purification steps do you think should be included in your table? Why do youth ink tha t ea ch of the va lues listed above is importa nt ? (Wha t a re the values telling

you a bout your purificat ion procedure?)

Your t a ble should include, at minimum , the columns list ed below.

Step Act iv it y(nm ol/m in

/l )

Pro te inconc .

(mg /m l)

Totalvo lume

( m l )

Totalprotein

( m g )

TotalAct ivi ty

(m ol/m in )

Specif icac t iv i t y

(m ol/m in /m g )

FoldP u r i f i -ca t ion

Yie ld(%)

B a sed on your da ta , wa s your purifica tion successful or un successful? Why? D o youha ve a ny da ta other tha n fold-purification to indica te wh ether your purification wa ssuccessful? How did your purification compare to literature values obtained forsimilar proteins? How did y our purifica tion of the mut a nt compa re to the purifica tionof the w ild-ty pe?

Enzyme assay: w ha t can you learn from each enzyme a ssa y? (If the a nswer isnothing, is it w orth including these results in the paper?) H ow do you know th a t t heassay results are valid? What assumptions are you making about the enzymerea ction a ctua lly occurrin g in th e reaction tube? Are th ese a ssumpt ions likely to becorrect for each a ssa y? Are th ese assum ptions l ikely t o be correct for some a ssa ysbut not for others? Wha t contr ols did you run t o ensure tha t t he results w ere a t leastpotentia lly mea ningful?

In some ca ses, the a nsw ers to the a bove questions do not n eed t o be stat ed explicit ly.However, you always need to consider the answers before writing the paper.Knowingly incorpora ting t he results of a fla wed experiment in a paper is a good w a y tolose grant funding or become unemployed, and may result in your finding yourself incourt defending yourself in a law suit or in a cr iminal t r ia l . This does not mea n t ha texperiments t ha t la ter tu rn out to be less informa tive tha n you would like are useless,but merely mean s tha t you should look ca refully at your dat a , and t ry to understa ndthe va lidity of each experiment before mentioning it in a wr itten document.

Scientific research involves intell igentobservat ion. In oth er w ords, you need to look atyour dat a cr i t ica lly , and t o a t t empt t o understa nd everyt hing i t is tel l ing you. Once

you believe tha t you underst a nd t he da ta , you need to describe the results of yourexperiments so tha t others w il l be able to a pprecia te your insights, a nd be able todecide whether they agree with your conclusions.

Discussion: This section should begin w ith a brief summa ry of your results, a nd a nexplana tion of wh a t t hey mea n. Wha t w ere you hoping to a ccomplish? Wha t did y oudiscover a s a result of your experiments? Which of your result s a re interest ing? Wha t


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ca n you say a bout your hypotheses now t ha t you have add itiona l dat a ? Wha t did youexpect t o see? Did y ou see wh a t y ou expected? Did you find sur prising result s?

At leas t in pa rt , the Discussion section should be th e section in w hich you a nsw er thequestions you ra ised in the In troduction. Sometimes the answ er is tha t your originalhypothesis turn ed out t o be fla w ed; in t his case, you should point out h ow your da ta

indicate the flaws, and propose a brilliant new hypothesis to account for yourobservat ions. S ometimes your origina l hypoth esis is supported by the da ta , in w hichcase you point out how your origina l brillian t concept predicted your results.

You sh ould end your discussion section w ith your conclusions. D id y our experimenta chieve your goals? How a re your results going t o cha nge th e world?

Figures and figure legends: In w rit ing a paper , f igures ca n be extremely useful.They a re ra rely, however, self-explan a tory. This mea ns t ha t y ou need t o refer to th efigure in the text. In a ddit ion, you need to include some releva nt informa tion in th efigure legend, so th a t people simply glancing t hrough th e pa per ca n derive usefulinformation from the figures.

As a n exam ple, in a f igure of a gel, you should indicate t he ident ity of th e sam plesloa ded in the figure legend. If more tha n one ba nd is present in a n importa nt lan e, it isoften a good idea to highlight th e importa nt ban d in some wa y. (Note: in doing so, donot w rite on t he actua l lane; instead, place an a rrow or other ma rker beside the gel, orbeside th e lane.)

Designing figures requires considera ble th ought. Wha t point a re you try ing to ma kewit h t he f igure? Is t he point necessary? If t he point is a necessary one, how ca n t hefigure be used to ma ke th e point a s clear a s possible? Ca n you design a f igure t opresent more informa tion, or present t he informat ion m ore clearly?

Figure legends can be extremely useful in allowing you to present relevantinforma tion th a t w ould disrupt t he orderly flow of idea s in th e text. The figure legendsa re a lso necessary in cla rifying th e informa tion presented in t he figure.

References: In a ny scholarly endeavor, it is customar y t o give credit t o your sourcesof informa tion. The Reference section a llow s you t o properly credit t he origina tors ofth e informa tion you a re presenting. Where did your int roductory informa tion comefrom? Where did your methods come from? (Note that, unless you invented themethod, you should alw a ys reference th e pa per t ha t first described the w ork.)

Acknowledgments: In scientific papers, it is customary to thank the agency,

compan y, or privat e founda t ion tha t fund ed the resea rch. In a ddit ion, it is poli te toacknowledge gifts of reagents or other supplies. Note that, if you purchased therea gent, t he source of th e reagent should be cited in t he Methods section.

Appendix: Fina lly , the report should conta in an a ppendix tha t conta ins your ra wdat a an d th e ca lculat ions tha t you used to reduce your da ta to understa nda ble form.In a real paper, Appendix sections are only included for the description of novel


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calcula tions; in th is course, th e Appendix is included so tha t y our la b instr uctor cancorrect your calculation mistakes.

In each section, at tem pt to orga nize the informat ion you a re presenting logically.Scient ific papers a re w ritt en for int elligent people who ha ve not done the experimentsyou are describing. If your report is disorgan ized th ey ma y not und ersta nd it. I f you do

not write well, the reader will not believe your conclusions. (In the real world, a poorlyw ritt en paper w ill not be published, and you will not get gra nt funding! In this class, ifyou inst ructor does not believe your conclusions, y ou will n ot get a good gr a de.)

The list of qu estions below is design ed to help you wr ite ea ch section of th e reportcorrectly.

Criteria for J udging Lab Reports:General:

Does it conta in t he required sections?Is it clear ly writt en?Does it use scientific terms properly?Does it use good gra mma r?Are t he w ords spelled correctly ?Are the calculations performed correctly?Is it unnecessa rily long?Is t he title mea ningful?Does th e tit le pa ge conta in the a uthors na me and a ddress?Does the title page conta in th e nam e(s) of the a uth ors lab pa rt ners?

Abstract:Does it int roduce th e overa ll topic?

Does it explain th e hypoth esis being tested?Are th e importa nt methods described?Does it rea ch logica l conclusions supported by t he da ta ?Does it flow w ell? Is it logica lly w ritt en? Is it concise?

Introduction:Does it give genera l background?Does it point out poorly un derstood or unknown factors relat ed to th e study?Does it ra ise questions?Does it explain th e hypoth esis being tested?Does it discuss th e significa nce of th e work?Does it flow w ell? Is it logica lly w ritt en? Is it concise?

Materials and Methods:Could th e experiment s be understood based on the informa tion given?Does it include the source of th e reagent s?Does it include informa tion th a t belongs in th e Results section?Does it describe a ll of th e methods used?Is it excessively long?


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Results:Does it expla in th e rat iona le and st ra tegy for the experiments performed?Does it describe, in words, wh a t w a s done?Does it a nsw er the questions ra ised in th e Intr oduction?Does it flow w ell? Is it logica lly w ritt en? Is it concise?

Discussion:Does it summa rize the findings obtained in t he Results section?Does it discuss the expected results?Does it discuss t he unexpected results?Does it a nsw er the questions ra ised in th e Intr oduction?Does it reach conclusions?Does it expla in w hy t he conclusions ar e importa nt ?Does it flow w ell? Is it logica lly w ritt en? Is it concise?

Figures:Are th e figures well designed?Do th e figures include informa tive legends?Do th e figures present informat ion useful for und ersta nding t he text?

Tables:Are th e ta bles well designed?Do the ta bles present informa tion useful for understa nding t he text?Is th e informa tion in the ta bles redundan t?

Acknowledgments:Are t he sources of funding given credit?

References:

Is t he informa tion obta ined from published sources properly referenced?

Appendix:Are the raw da ta a nd th e ca lculat ions included?

Rea ding over th is list of quest ions before writing a dra ft of th e report is st ronglyrecommended. Read ing these quest ions a f ter wr it ing your f irst d ra ft , a nd using t hequest ions t o guide your revisions is a lso strongly recommend ed.


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Basic Laboratory Techniques: Pipetting

In molecular biology a nd biochemist ry, t he a bility to a ccura tely an d reproduciblymeasure a nd t ra nsfer sma ll volumes of liquids is crit ica l for obta ining useful results.For volumes less tha n 1 ml, t he most common meth od for mea suring liquid volumesinvolves th e use of a device know n a s a pipetma n. (Note: P ipetm a n is th e bran dna me of t he most comm only used of t hese t ypes of pipets; however, a ll of t hesepipett ing d evices w ork on simila r prin ciples.)

A dra w ing of a pipetma n is shown a t r ight. The devicesyou use ma y not look exactly like th e one show n. Thepipetmen used in th is cours e come in thr ee differentty pes: P 1000, P 200, a nd P 20.

P 1000 a re useful for volum es from 200 t o 1000 l.P 200 ar e useful for volumes from 20 to 200 l. P 20 a reuseful for volumes from 0.5 t o 20 l. Ma ke sure tha t

you ar e using t he correct pipetma n for th e volume youneed. Also, ma ke sure tha t the pipetma n is a ctua lly setfor the volume you need by looking in the volumewindow, and, if necessary, turning the volume controlknob unt i l th e pipetm a n displa ys t he correct volume(th e pipetmen do notread y our mind; because severa lpeople w il l use th e pipets, t hey ma y n ot a lwa ys be setas you expect them to be). Do not attempt to setpipetmen for volumes larger than theirmaximum, or for volumes less than zero; doing sowill dama ge the pipetma n.

All pipetmen use disposable tips (do not pipet liquids without using theappropriate tip, beca use this wi l l conta mina t e the p ipetma n a nd ma y da ma ge it ).When a tt a ching t he tip, make certa in tha t the t ip is the correct type for thepipetman you are using, and that the tip is properly seated on the end of thepipetman.

Try depressing t he plunger. As t he plunger depresses, you w ill feel a sudden increasein resist a nce. This is th e first st op. If you continu e push ing, you will find a pointw here the plunger no longer moves dow nw a rd (th e second stop). When using th epipet, depress the plunger to th e first st op, pla ce th e tip into the liquid, a nd in aslow, controlled manner, a llow th e plunger to move upwa rds. (Do not simply let t heplunger go; doing so will cause the liquid to splatter within the tip, resulting in

ina ccura te volumes an d in conta mina tion of the pipet.)

Now , ta ke the pipetma n (ca rrying t he pipet ted l iquid in th e t ip) to the conta iner towh ich you w ish to a dd liquid. Depress th e plunger to the first , a nd t hen to th e secondstop. If you wa tch carefully, you will note tha t d epressing to th e second st op expels allof the liquid from th e tip. (Actua lly, this is t rue for most a queous solutions. In somecases, however, such as for organic solvents, or for solutions containing largea mounts of protein, it is often difficult t o get a ll of th e liquid out of the tip. In t hese


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cases, it is best t o wet t he tip, by pipett ing t he original solution once, expelling it ,an d th en ta king up the liquid a second t ime.)

Although pipetmen a re tremendously useful, they h a ve a potentia l dra wba ck. If usedimproperly, pipetmen will tra nsfer inaccurat e volumes. In addition, pipetmen ma ylose calibr a tion. If used incautiously, t herefore, pipetmen ma y yield misleading or

even totally useless results. Checking the calibration of pipetmen is a simpleprocedure tha t can sa ve considera ble time, energy, a nd rea gents.


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Basic Laboratory Techniques: Measuring Absorbance

A spectrophotometer is a n inst rument for mea suring t he a bsorbance of a solut ion.Absorba nce is a useful qua ntit y. The Beer-La mbert law sta tes tha t :

A = cl

where A is th e a bsorban ce of the sample a t a par ticular wa velength, is th eextinction coefficient for t he compound a t th a t w a velength in (M cm )-1, cis the molarconcentra tion of the a bsorbing species, an d lis the pa th length of the solution in cm.Thus, if t he extinction coefficient of a n a bsorbing species is known , th e ab sorban ce ofth e solut ion can be used to ca lcula te t he concent ra t ion of the a bsorbing species insolution. (This a ssumes th a t t he species of interest is th e only ma teria l tha t a bsorbsat the w avelength being measured.)

The above is an explanation of whywe mea sure absorbance: a bsorba nce a llow s us tocalculat e the concentr a tion of compound s in solution. How ever, it does not explainwhata bsorbance is. Another d efinition of a bsorba nce is:

A = log

I0

I

where I0 is the amount of light

entering the sample, and I is theamount of light leaving the sample.Absorbance is therefore a measureof th e portion of the light lea ving th elamp tha t a ctua l ly ma kes i t t o thedetector. A little thought will revealtha t w hen absorban ce = 1, only 10%of th e l ight is rea ching the detector ;w hen a bsorban ce = 2, only 1% of the

light is rea ching the detector. The typica l interna l arr a ngement of aSpectrophotometer

Absorbance values greater than 2 are unreliable, because too little light isreaching the detector to allow accurate measurements. When measuringa bsorba nce, note the va lues; i f the rea ding is greater t ha n 2, dilute the sa mple a ndrepeat t he mea surement.

Spectrophotometers measure the decrease in the amount of light reaching thedetector. A spectr ophotometer will interpret fingerprin ts on the optical face of th ecuvette, or air bubbles, or objects floating in your solution as absorbance; you

therefore need to look carefully at your cuvette before putting it into thespectr ophotometer to ma ke sure tha t y our readin g is not subject to these types ofar t i facts .

Cuvet t es ar e usually sq ua re objects 1 cm a cross (a s shown in th e above figure). Insome cases, the liquid reservoir is not sq ua re; in those ca ses, make sure th a t t he 1 cmdimension is a ligned w ith t he light path (note the orientat ion in the dia gra m a bove.)


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Some cuvettes a re designed for visible light only. When t he spectr ophotometer is setfor ultr a violet wa velengt hs (wa velengt hs of 340 nm or less) make sure that yourcuvette does not have a large absorbance when it contains only water.

The t erm spectr oscopy comes from t he w ord spectr um w hich originally referred tothe multiple colors of light apparent in an analysis of white light using a prism.Spectroscopy therefore implies the use of multiple wavelengths of light.Spectrophotometers ha ve the a bil ity to specif ica lly mea sure a bsorba nce at specif icwavelengths. The most commonly used method to allow this involves amonochroma tor, a device (either a prism, or m ore commonly, a diffraction gra ting)tha t splits th e incident light into its component w a velengths, a nd a llow s only light ofth e desired wa velengt h to reach th e sample. The abil i ty t o mea sure absorba nce a tdifferent wavelengths is very useful, because the extinction coefficient of acompound varies with wavelength. In a ddition, the a bsorba nce spectrum of acompound can vary dramatically depending on the chemical composition of thecompound, and depending on the environment (such as the solvent) around thecompound.

The graph at right showsthe absorbance spectrumof a protein. The proteinhas a s t rong absorbancepeak near 280 nm, butexhibits very littleabsorbance at longerwavelengths. For thisprotein, the onlychromophores (chemicalgroups with in a compound

th a t a bsorb light) ar e thearomatic amino acidstryptophan and tyrosine.For many proteins, thesetwo residues are the only chromophores; because tryptophan and tyrosine onlyabsorb in the ultraviolet portion of the spectrum, such proteins are colorlessmolecules. Colored proteins, such as hemoglobin, exhibit their color due tochromophores (heme, in t he case of hemoglobin) tha t a bsorb in t he visible port ion ofthe spectrum.

The extinction coefficient of a molecule at a given w a velengt h ca n be calculat ed usingth e B eer-La mbert equa t ion from absorban ce measur ements for solut ions of know n

concentration.


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Basic Laboratory Techniques: Performing Dilutions

Many solutions used in biochemistry are prepared by the dilution of a moreconcentra ted st ock solution. I n prepar ing t o make a dilution (or series of dilutions),you need to consider th e goa l of th e procedur e. This mea ns t ha t y ou need t o considerboth t he desired fina l concentra tion an d required volume of the diluted ma teria l. Asimple equa tion a llow s th e dilution to be ca lcula ted rea dily:

C 1V1 = C2V2

where C1 is the concentra tion of th e init ia l solution; V1 is th e volume of th e init ia lsolut ion ava ilable to be used for dilut ion (th is may n ot be the tota l volume of theinitial solution, a nd inst ead ma y be a sm a ll fraction of the initial solution), C2 i s t h edesired final concentration, and V2 is th e desired fina l volume.

In m ost ca ses, the initia l concentra tion an d th e fina l concent ra tion ar e either knownor ar e chosen in order to w ork correctly in t he experiment b eing plan ned. The fina l

volume is usua lly an a mount th a t is chosen based on the a mount required for a givenexperiment. This mean s t ha t a t least thr ee of th e required t erms a re either known orcan be chosen by t he experimenter.

Let us consider a n example. You a re set t ing up a sta nda rd curve. You ha ve a st ocksolution of 1000 g/ml B S A, a nd for one of th e point s on t he curve, y ou w a nt 200 l of20 g/ml. In this ca se, C 1 = 1000 g/ml; C2 = 20 g/ml, a nd V2 = 200 l. This lea ves V 1as t he unknow n va lue (i .e. how m uch of th e stock solut ion mus t b e dilut ed t o 200 lfina l volume to yield th e desired concentra tion). Rearra nging th e dilution equat iongives:

V1= V

2 C1

C2

a nd th erefore ( )4 l = 200 l20 g/ml

1000 g/ml

Thu s, y ou need t o dilut e 4 l of th e st ock solut ion t o a fina l volume of 200 l (i .e. byadding 196 l).

If, in t he exam ple, you w ished t o ma ke a solut ion of 1 g/ml, th e sa me equa tion w ouldind icat e th a t you n eed 0.2 l of t he 1000 g/ml s t ock solut ion for 200 l of th e fina ldilut ed sa mple. This is a problem: 0.2 l is very difficult t o measu re a ccura tely. Youha ve two choices: cha nge the f inal volume (i .e. if V2 is larger, then V1 must a lsoincrease), or perform serial dilutions (i.e. instea d of diluting th e stock solution by afa ctor of 1000 in one step, dilut e the st ock solut ion, a nd t hen m a ke a furt her dilut ionof the diluted stock).

In m an y cases, wh ile the fina l concentrationis importa nt, th e fina l volumeis not (a s inth e previous para gra ph). In t hese ca ses, do w ha t w a s explained in th is exam ple: use aconvenient dilution: a dilution that involves volumes that are easily pipetted.P ipett ing 1.3333 l is usua lly less a ccura te t ha n pipettin g 4 l, both because 4 l is ala rger volume, a nd beca use it is d ifficult t o set t he pipet for 1.3333 l. In t his ca se, 4l is a conven ient volume, w hile 1.3333 l is n ot.


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In some ca ses, you may not know t he a ctua l sta r t ing concentr a t ion. I f , for example,you need to measure th e enzyme act ivity in a sam ple, a nd you f ind t ha t t he act ivityis too high to measure a ccura tely, you w ill need to dilute the sta rting m a terial. Sinceyou dont know th e actua l sta r t ing concentr a t ion, a l l you know is the concentr at ionratio between starting and final solutions. As long as you keep track of theconcentration ratio in all of your dilutions, you can easily determine the enzyme

a ctivity in th e initial solution, even t hough you ca nnot mea sure it directly.

Concentra t ion ra t ios ar e frequently of considerable va lue. For example, you h a ve ast ock solution of buffer th a t conta ins 450 mM Tris-H C l, 10 mM E DTA, a nd 500 mMNa Cl. You a ctua lly w ish to use a final concentra tion of 45 mM Tris-HC l, 1 mMED TA, an d 50 mM Na Cl. In each case th e concentr a t ion of th e f inal buffer is one-tent h t ha t of th e origina l. Simply performing a 1:10 dilution of th e stock solution th engives th e appropria te fina l concent ra tion of each component . The st ock solution ofbuffer is ty pica lly called a 10x stock, beca use it is ten-times m ore concent ra ted t ha nth e fina l, useful buffer.

Note, in the previous paragraph, the 1:10 dilution. The description uses thechemistry convent ion for th is term , wh ich w ill be used th roughout t his course. The1:10 dilution mentioned is performed by ta king one part of the initia l solution, a nda dding nine par ts of solvent (usua lly wa ter). This results in a fina l concentra tion tha tis ten-fold low er tha n t he origina l.


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Basic Laboratory Techniques: Buffers

Proteins, and especially enzymes, are generally quite sensitive to changes in theconcent ra tions of various solution components. A buffer is a solution th a t is used t ocont rol the propert ies of a process occurring in a n experimen ta l a queous medium . Theterm buffer is related to the ability of these solutions to resist changes in thehyd rogen ion concentra tion, but buffers a lso cont a in other molecules, and a re used toattempt to influence the ionic strength, the activity of proteases, and otherpara meters of the experiment in a ddition to the h ydrogen ion concent ra tion.

In a ny biochemist ry experiment , the buffer component s must be chosen based onth eir effect on t he experiment. I dea l buffer component s cont rol pH a nd ionic strengt hw ithout intera cting in oth er wa ys w ith th e system being studied. For example, w hilephospha t e is a common physiological buffer, it m a y not be a ppropriat e for somebiochemical experiments, especially if phosphat e is a subst ra te or product of t hereaction being studied. In ad dition, some proteins int era ct poorly w ith some buffercomponents (a fa ct usua lly discovered by t ria l a nd err or). As a n exa mple, Tris is less

th a n ideal beca use of its high pKaa nd th e la rge cha nge in pKath a t i t exhibits uponcha nges in temperat ure. However, Tris is inexpensive, most proteins ar e st a ble inTris buffers, an d Tris rar ely rea cts with biologica l compounds; a s a result , Tris iscommonly used in biochemistr y.

You w ill ha ve seen th e Hend erson-Ha sselbalch equat ion in previous courses. Thisequat ion is useful for calcula ting t he theoretical pH of a solution. It is a lso useful forpredicting w heth er a par ticular compound w ill be useful as a buffer over a given pHrange. However, the Henderson-Hasselbalch equation has its drawbacks. Manybuffers used in biochemica l experiment s deviat e significa nt ly from ideal Henderson-Ha sselbalch beha vior.

pH = pKa+ log[A ][H A ]

Henderson-Ha sselbalch equa tion

Because of the commonly observed deviations from ideal behavior, buffers arety pically prepa red by adding th e buffer component s to a cont a iner, adjusting thesolut ion to th e desired pH by a dding a n a cid or a ba se, and then adding sufficientw a ter t o rea ch the expected fina l volume.

For example, a 1 liter of 50 mM Tris-HCl buffer (pH 7.4) with 200 mM sodiumchloride w ould be prepa red by ad din g 50 mm oles of Tris ba se and 200 mmoles of

sodium chloride to a flask a nd a dding w a ter to about 900 ml. HC l would then be add edto reduce the pH to 7.4, using a pH meter t o monitor t he chan ging pH, follow ed bya ddition of enough w a ter t o yield a 1 liter fina l volume. (If t he solution cont a ined 1 literbefore ad dition of th e HC l, the fina l volume would be more tha n 1 liter, a nd t hereforethe buffer w ould be less concent ra ted t ha n it should be.)

Note th a t in order t o produce most biochemica lly useful buffers, severa l component smust be added together. This frequently requires careful consideration of thenecessar y d ilutions for each of th e component s.


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When performin g experiments w ith proteins, it is ra rely a good idea to dilute th eprotein with water, unless denaturation of the protein is not a concern. Whendena tur a tion is a concern, such a s when performing dilutions for enzyme a ssa ys ,perform t he dilution using a suita ble buffer t o prevent undesirable alt erat ions in t he

str ucture of the protein in solut ion.


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General Information: Molecular Biology

Purifying a protein from cells that normally contain the protein has certainad van ta ges. You know t ha t t he protein wa s synth esized in i ts na tura l environment ,a nd you ca n a ssume tha t it w a s folded correctly, a nd w a s subject t o a ny norma l post-translational modifications. Purifying proteins from normal cells has been thetra ditional m ethod for most of the hist ory of biochemistr y.

However, using normal cells as a source of protein a lso ha s signif ica nt dra w ba cks.The proteins suit a ble for st udy a re limit ed to those expressed by readily a vaila blesources, and to those expressed in significant quantities. For example, studyinghuma n proteins tends to be somew ha t dif f icult due to l imita t ions in ava ilabili ty ofsta rting ma terial. In a ddition, the experimenta l modifica tions tha t can be intr oducedint o th e protein ar e limit ed both in ty pe a nd in specificity .

As a result , expression of genes in heterologous organ isms (especially ba cteria) ha sbecome a frequent ly used technique. Ba cteria l protein expression genera lly a llow s th e

protein to be expressed in very lar ge qua nt it ies, a l low s th e resea rcher to chose theform of the protein to be expressed, an d a llow s th e resea rcher to introduce mut a tionsin th e protein sequence to exam ine the roles of individua l a mino a cids in t he functionof the protein.

Escher ichi a colii s a ba cter ium found in th e intes t ina l t ra c ts of a ma ny species ofmammals, including humans. It has been used for a wide variety of molecularbiologica l experiments, a nd a lar ge number of specia lized la bora tory E . colis t r a insha ve been produced. Techniq ues for m a nipulat ing D NA in E. colia re well esta blished.

Comments on molecular biological techniques

Molecula r biology in its m odern mea ning (i .e. referring to genetic ma nipulat ion a nda na lysis techniq ues) is a fa ir ly new science; nearly a l l of the techniques used wereinvented a fter 1970. This mean s tha t t echniq ues are still being invented, an d ma ny ofth e procedures ha ve cha nged (often dra ma tically) during t he last few y ears.

Molecular biological techniques differ slightly from biochemical techniques. Inmolecular biology, exact quantitation of the reagents is of variable importance. Insome cases (as with some of the buffers) concentrations and pH are criticallyimport a nt ; in others, such a s ligat ion reactions, t he D NA concent ra tion can va ryw ith in a fa ctor of ten (or more) a nd s till a llow th e procedure t o be successful.

On the other hand, some aspects of molecular biology require considerable care.H uma ns (an d m an y o ther o rga n i sms ) sec rete enzymes th a t deg r ad e DN A an d

RNA ; it is t herefore necessary to be completely a w a re of wha t you ar e doing. Allow ingth e reagent s t o conta ct your skin w ill ca use problems not necessa rily to you, butdefinit ely to your sa mples. DNa se (th e non-specific enzym e tha t degr a des DN A) isra pidly denat ured by heat ing a t 68 C; for this reason, i t is a good idea to hea t t reatyour DNA sa mples if there is a ny cha nce of DNa se cont a mina tion. Your plast icw a re(th e pipet tips and microfuge tu bes) ha ve been a ut ocla ved (heat ed to 121 C a t


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eleva ted pressure) to dena ture any D Na se a ssocia t ed wi th them; this means tha tyou should not ha ndle the pla sticwa re unnecessarily (e.g. , pour out a few m icrofugetubes; do not reach int o the cont a iner wit h y our fingers).

Ma ny molecular biological procedures a re very comm only used. Some techn iques a reso commonly used tha t a l l of th e required rea gents a re a vaila ble in kit form. These

kits ha ve mad e man y a spects of molecula r biology much easier.


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General Information: Cell Genotypes

A lar ge var iety of E . colist ra ins a re used in molecular biological resear ch projects.The str a ins a re usua lly different ia ted by t heir genetic properties. Resear chers choosethe strain or strains that they will use for an experiment based on the geneticproperties of the str a ins, an d th erefore need some understa nding of the genes tha t a refrequently modified in la bora tory str a ins of E. coli.

A genotype for a n E. col istr a in includes the genes tha t a re know n to be modifiedwhen compared to the wild-type strain from which the strain was derived. Mostlabora tory str a ins a re derived from either K12 or B str a ins, which a re considered tobe sta nda rd w ild-ty pe stra ins. Genes not listed in a genoty pe a re thought not to bemutated, although laboratory strains often deviate from wild-type in additional(poorly cha ra cterized) w a ys. In most cases, the genes listed a re muta ted t o the pointof ina ct iv i t y . In a few cases , a - (indicating inactive or absent), + (indicatingactive), or (indicat ing deleted) is a dded to a void a mbiguity.

The genotype is a fa ctor in choosing a ba cteria l stra in. However, once a str a in ha sbeen tent a t ively chosen, i t usua lly has t o be test ed to verify i ts usefulness in theexperiment.

G enotypes for t w o commonly u sed E. colistra ins are shown below.

Top10 genotype: F -, mcrA, (mr r-hsdRMS-mcrB C )80l acZM15 l acX74deoRr ecA1 ar aD139 (ara-leu)7697 ga lU ga lK r psL endA1 nupoG

J M109 genotype: F t r aD36 lacIq lacZM15 proA+B+/ e14-(McrA -)( lac-proAB) th igyr A96 (N alr) endA1 hsdR17(rK- m K+ ) r elA 1 supE 44 recA1

F = a low copy number plasm id (a lso called an episome) th a t can be tra nsferred fromone cell to an oth er . The genes on t he F plasmid a re l isted immediat ely a f t er this ;genes after t he slash a re genomic.

F - = cells tha t la ck th e F plasmid. This plasm id w a s of considera ble concern du ringth e ea rly yea rs of recombina nt DNA experimenta tion, because it a llow ed bacteria t oexcha nge DNA with out the a ssistan ce of th e resea rcher.

lacZM15 = A mut a ted form of lacZ(-ga la ctosida se) missing th e first 15 codons. C o-expression of lacZ , a gene for a sh ort pepti de (of 41 t o ~ 150 residues) results inrestorat ion of -ga lactosida se a ctivity.

lacIq= a modified form of the lacIrepressor gene. In t his case, the muta tion results inover-production of the LacI protein, and therefore greater suppression of lac promoter(and lac-derived promoter) driven expression.

e14= a D NA element present in K12, but n ot ma ny deriva tive stra ins. The elementconta ins the McrA gene, so cells la cking e14a re also lacking McrA .


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mcrA, mcrB C, mr r= genes for methy lat ion-requiring restriction enzymes. DNA withmethylation is degraded by these enzymes; DNA molecules isolated from higherorganisms and from some non-E. colibacteria (although not from DrosophilaorSaccharomyces), conta in met hyl gr oups a t sit es recognized by these enzym es. (Notetha t t his only a pplies to DNA isolat ed directly from the organism; P CR products a ndother i n v i t rosynt hesized DNA molecules are not meth yla ted.)

proA+B+= cont a ins a ctive forms of tw o genes required for proline biosynt hesis. Thesegenes ar e sometim es used as a select ion mecha nism. I n J M109 cells , th e episomeconta ins the proABgenes, wh ile the chromosoma l DN A is proAB. If J M109 cellsa re grown on a cultur e medium la cking proline, only th ose cells ha rboring t he episomew ill be a ble to survive.

endA = E ndonuclea se I; deleting t his gene reduces non-specific degra da tion of DNA.

r ecA1 = recombinat ion nega tive, a mut a tion genera lly necessar y for sta ble replica tionof plasm id D NA.

hsdR = deletion of th e norma l K12 restriction enzym e EcoKI . The genoty pe hsdR17(rK- m K+ ) means tha t the res t r ict ion enzyme is de le ted, but t ha t the methyla t ion isreta ined; this allow s DNA replica ted in t his stra in to be used in hsdR + s t ra ins .

hsdRMS = deletion of both EcoKI restriction enzyme and the correspondingmethylase. This means that DNA produced in this strain may be degraded iftra nsformed into a non-hsdRMS st ra in .

supE= gluta mine-inserting a mber (U AG ) suppressor tRNA.

supF= ty rosine-inserting a mber suppressor t RNA. The supEa nd supFsuppressors

a re requir ed for growt h of some bacteriophages. The presence of supEa nd supFinmany laboratory strains means that use of TAG stop codons may result inunpredicta ble results, a nd th erefore, w hen possible, th e oth er st op codons (TAA orTGA) should be used.


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Methods: Polymerase Chain Reaction

P olymerase cha in react ion (P CR) is a t echnique th a t a l low s the generat ion of largeamounts of a single DNA sequence from a mixture of sequences; the fragmentgenerat ed ca n be designed to conta in specific sta rtin g a nd ending positions ba sed onth e needs of th e experiment.

P CR u ses a D NA polymera se (a n enzyme th a t syn th esizes DNA). DNA polymera sesrequire a s igna l to begin synt hesis . The s igna l is a shor t f ra gment o f DNA. P CRuses tw o synt hetic oligonucleotides (known a s primers) tha t correspond to (a nd th usba se-pa ir to) th e ends of t he sequence of int erest and a ct a s synt hesis init iationsignals.

If t he C ycle 1 reaction (th e top ofthe figure at right) were theentire process, PC R w ould not bevery useful. However, an

examina tion of the figure revealstha t , a t t he end of the first cycle,twice as much template DNAexists as was present a t thebeginning. Therefore, repeatingthe cycle allows the amount ofproduct DNA to increase geo-metrically.

In th eory, t he a mount of productwill double each cycle. Inpractice, PCR is not quite that

efficient, a lth ough it can producetremendous quantities of DNA.It is literally possible to beginwith a single molecule of DNAand generate enough DNA forany molecular biologicaltechnique. In addition, the DNAsynt hesized in the P CR r eact ionwill have specific starting andending points: the primersequences define th e ends of thefragments.

The tw o st ra nds of the DNA templa te ar e separa ted byheating (usually to 94C). The temperature is thendecreased to allow the primers to bind to the templateDNA. Once the pr imers ha ve bound , the polymerase i sa l lowed to syn th es ize new D NA s t ra nds ( the polymeras e

most commonly used ha s a t empera tur e optimum of 72C .)

Good P CR primers cont a in approximat ely 50% G + C and 50% A+ T. This reducesproblems in inducing templa te stra nd separa tion ca used by the high a ffinity of G forC, a nd reduces non-specific priming common w ith high AT cont ent.


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In addition, good primers have few regions of complementarity either internal toprimer (i .e. secondary structure) or between two primers, especially at 3 end ofprimer, a nd a void repea ted sequences (e.g., AAAA or GTGTGT). Primers that arepoor by these criteria can ca use art ifacts t ha t prevent a mplificat ion of th e desiredsequence. In some cases, these potential problems are difficult to avoid due toconstraints imposed by the sequence of interest ; in these cases, the use of longer

primers (i .e. 24 bases) may solve specificity problems.

The primer should be long enough to ha ve a rea sona ble melting t emperat ure (i .e. amelting temperature of 55-70C). Melting temperature depends on a number ofvar iables. In m ost cases, an a pproximat ion [(4C for each G or C ) + (2C for each Aor T)] w ill yield a va lue close enough t o design t he P CR experiment.

P CR a lso a llows t he generat ion of mutat ions a t t he ends of the fra gment, beca use thepr imer does not need to be an exa c t ma tch t o the templa t e DNA (i t n eeds t o be asufficiently good ma tch t o allow primer binding, but it does not need to be a perfectma tch). In m ost ca ses, the intr oduct ion of misma tches a t th e 5 end of the primerha s lit t le effect . However, misma tches a t the 3 end of th e primer ma y preventsynth esis of the new stra nd.

The muta t ions inserted by P CR a re frequent ly used to genera te restr ict ion si tes tosimplify cloning of the P CR fra gment . Although P CR can a lso be used to genera temut a tions w ith in coding sequ ences, th is is somewh a t more complex, becau se t heP CR primers only a f fect the sequence at t he ends of th e PC R fra gments. How ever,modified forms of P CR a re quit e useful for sit e-directed mut a genesis experiments.

P CR requires th e use of a DNA polymera se to ma ke the copies of the D NA sequenceused as a template . As noted in th e f igure above, the P CR method involves hea t ingthe sa mple to ~ 94 C t o separa te the chains of the double-stra nded DNA. In a ddition,

w hile the oligonucleotide binding a nd polymerization rea ctions can occur at a ra nge oftem pera tu res, oligonu cleotide bind ing is m uch more specific (i .e. it is m ore likely th a tth e oligonucleotide will bind th e correct seq uence) a t h igher t empera tu res. To preventproblems w ith low t empera ture incubations an d problems due to denat ura tion of thepolymera se, most P CR experiment s employ thermosta ble D NA polymerases. TheDNA polymerase most commonly used for PCR is derived from the bacteriumTh ermu s aquati cus. T . aquati cusprefers to live a t t empera tur e of about 70 C, a ndtherefore its proteins (including its DNA polymerases) are stable at elevatedtemperatures.

Although t he Taqpolymerase is highly thermostable, it does begin to denature attemperatures above 90C. Its half-life decreases rapidly with increasing

tempera ture. For this rea son, melt ing t imes of great er tha n 1 minute for t he cha inreact ion cycles should be a voided. I f th e therma l cycler m elt ing temperat ure drif tsa bove 95 C, th e enzym e may be ina ctivat ed prior to completion of the progra m.

The Taqpolymera se ha s a primer extension r a t e of 60-100 ba ses/second underoptimum conditions; thus it may be advantageous to use short (1-10 second)extension times, particularly for short products (i .e. below 500 bp) to decreaseforma tion of non-specific products. H owever, for longer fra gment s (grea ter t ha n 1000


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ba ses), optim um primer extension rat es a re ra rely a chieved, and longer extensiontimes sh ould be used.

Although t he Taqpolymerase is a popular enzyme due to its ability to catalyzeprimer extension under a w ide variety of conditions, it ha s some dra w ba cks. Its w orstdra w ba ck is its rela tive lack of fidelity; it ha s a significa nt error ra te, and la cks

proofrea ding functions. As a result , man y experiment s employ th ermosta ble DNApolymerases that have lower probabilities of misincorporation. The use of otherpolymera se ma y r equire minor modifica tions t o the P CR procedure described below.

P CR condit ions, a nd especia lly an nea ling temperat ure, must be chosen empirica lly tooptimize P CR product forma tion. Some primer-templat e combina tions w ork under awide variety of conditions; others result in product only in a narrow range ofconditions. For some reactions the annealing temperature is a crit ical parameter,w ith no specific product formed except in a na rrow optimum r a nge. It is usua lly bestto begin testing conditions w ith a n a nnealing t emperat ure of a bout 55C , beca use theuse of an nea ling tempera tur es above 50 C often prevents cert a in ty pes of misma tchart i facts .

The tempera tur e profile shown below is a t ypica l one th a t w orks for ma ny d ifferentprimers an d templates:

PCR procedure: PCR Temperature Profile

Mix reagents in a P CR t ube: Temp. Time Function16 l of mixture of 1.25 mM dNTP 94 C 0.5 minut es D ena t ure D NA10 l 10x P C R b uffer5 l 20 M 5 primer

55 C 0.5 minut es P rimerAnnealing

5 l 20 M 3 primer1 l of DNA templa te m ixtur e

62.5 l deionized w a ter

72 C 0.75 minut es P rimerExtension

0.5 l TaqpolymeraseP lace the PC R tube in the P CR ma chine.

Run t he P CR progra m a s shown a bove (wit h a ppropriat e modif ica t ions i f necessaryfor the specific polymera se, primers, a nd t emplat e being used in t he experiment ).


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Methods: Plasmid Preparation

P CR is one method for genera ting lar ge am ount s of DNA. A second method is to a llowbacteria to replica te th e DNA and t hen purify the replica ted DNA from t he bacteria.In most cases, the D NA of interest for th is method is plasmid D NA. A plasmid is adouble stra nded DN A molecule tha t w ill replicate in a n organ ism. A typical plasmidused for molecular biology cont a ins a t least four fea tur es.

1) The plasmid must be circular, because bacteriagenerally w ill not replica te linea r D NA. 2) The plas midmust conta in a sequence tha t funct ions a s an originof replication (ori). 3) The plasmid must contain aselection mechanism th a t w i l l force th e ba cter ia t oreta in th e DN A; th e most common type of selectionmecha nism used in bacteria is a gene for resista nce toa n a nt ibiotic such a s a mpicillin. 4) The plasm id mustcont a in a region for th e insertion of th e experimental

DNA. A generic plasmid exhibiting these features isshown a t right.

An expression plasmid is a specific type of plasmid used to allow expression ofheterologous DNA. An expression plasmid must therefore have, in addition to thefeat ures listed above, a st rong promoter element th a t w ill drive tra nscription of th eforeign DNA1 in th e host organ ism, and a n effect ive r ibosome binding si te tha t w il la llow efficient tr a nsla tion of the tr a nscribed RNA.

B eca use plasm ids a re much smaller th a n chromosomal D NA (for E . coli, a t y p ica lplasm id cont a ins 5 to 10 kiloba se pairs (kb), wh ile E. colichromosomal D NA conta ins

about 4,800 kb), separating the two types of DNA molecule is relativelystra ightforwa rd. In a ddition, most pla smids used in molecula r biology a re high copynum ber plasm ids; in other w ords, each bacterium conta ins ma ny copies (usua lly>100) of the plasmid. Therefore, although each plasmid molecule is much smallerth a n t he chromosome, plasmid D NA often comprises ~ 10%or more of t he tota l DNAin the bacterium.

Plasmid preparation requires several steps:1) Growth of bacteria containing the plasmid of interest. This involves sta rt inga liquid culture usin g a single clone (each colony on a plat e represents a single clone,so cultures a re sta r ted by picking one colony a nd a dding i t to th e culture medium).The ba cteria a re th en grown unt il th ey reach sta t iona ry phase; sta t iona ry phase

occurs w hen th e bacteria ha ve either used up a ll of the ava ilable nutrient s, or w henthe ba cteria l w a ste products ha ve rea ched levels th a t preclude further growt h. Thecommonly used labora tory ba cterium E. coli, reaches s ta t ionar y pha se a f t er 12-18hours of growth (typically overnight growth is assumed to result in stationary phase).

1N o t e t h a t f or e i g n D N A c a n b e f r o m a n y or g a n i s m , i n c lu d i n g t h e o r g a n i s m u s e d a s a h o s t .Thus, E . col ican be used t o express E. col ip ro t e in s f r o m e xp re ss ion p l a sm id s . I n m o s t c a se s ,however, the D NA being expressed is f rom a dif ferent organism , a nd is being expressed in E . col ifor convenience.


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(Note tha t some other ba cteria l types, and a ll yeast a nd ma mma lian cells in culturegrow more slow ly th a n does E. coli.)

2) Separation of the bacteria from the culture medium. The next step has tw opurposes, concentrating the cells, and transferring them into a buffer that willfa cilita te plasm id purifica tion. The usua l method is to centr ifuge th e cultur e an d to

discard th e spent culture m edium. The cells can th en be frozen for s tora ge (freezingthe cells also increases the vulnerability of the cells to the lysis conditions), orresuspended immediately in a buffer for plasmid prepara tion.

3) Lysis of the bacteria (i .e. disr uption of th e ba cteria t o releas e plasmid DNA).B a cterial cells are much tougher t ha n hum a n cells, an d lysing bacteria requires someeffort . The techniq ue usua lly used to extra ct plas mid DNA from bacteria involveslysing the cells with a mixture of SD S a nd Na OH, w hich disrupts the cell membra nesa nd cell wa ll. Once the SDS/NaOH mixture has been added to the cells, it isimperative that the cells be treated gently; DNA molecules ar e long a nd fr a gile,and vigorous treatment will readily damage the DNA molecules released from thecells. In addition, when performed properly, the alkaline lysis technique does notextract the chromosomal DNA (unless the chromosomal DNA is fragmented byviolent shea ring forces). The int ention in a plasmid prep is t o purify t he plasm id DN Aw hile obta ining a s litt le chromosoma l DNA a s possible.

4) Neutralization of the NaOH. Once th e cells ha ve been lysed, it is a good idea tolower t he pH to n ear n eutral. Neutra lization is typically performed by adding a nacetate buffer. This has two effects: it prevents degradation of the DNA, and itprecipita tes some proteins a nd most of the lipids.

5) Purification of the plasmid DNA. A variety of different techniques a re used forsepara t ing plasmid DNA from t he other molecules present in th e cell. Most of th e

commonly used t echniq ues involve binding th e pla smid D NA to a n insoluble ma teria l,w a shing off unbound m a teria l (protein, lipids, an d sma ll molecules), an d th en elutin gthe D NA.

Procedure for Plasmid miniprep:The procedure out line given below should be used as a genera l guide for t he a lkalin elysis technique. However, because of minor differences in the pla smid prep kits incommon use, it is a good idea t o consult t he ma nufa cturers inst ructions for t he exactprocedure.

1. G row a 3 ml overnight culture of the cells cont aining the plasmid, using t hea ppropria te culture medium (usua lly either LB or TB cont a ining a n a nt ibiotic

to force the cells to reta in t he plasm id).2. Cent rifuge 1.5 ml of the culture in a microfuge tube t o pellet t he cells; disca rd

th e superna ta nt . Dependin g on t he size of th e cell pellet, it is frequent lypreferable to repeat the procedure by a dding a n a dditional 1.5 ml from thesa me cultur e to the pelleted cells, an d recent rifuging.

3. Resuspend t he cells in th e Resuspension B uffer solution (the a mount t o beused va ries from 200 to 350 l depending on t he kit). Make sur e tha t t he cells


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a re evenly su spended. (The sus pension should be cloudy w ith no obviousclumps).

4. Lyse th e cells using a mixture of SD S a nd Na OH (usually 1%SD S in 0.2 MNaOH 2. After add ing the SD S a nd Na OH, be gentle with the solution to a voiddisrupt ing t he chromosoma l DN A. The solution should become fairly clear .

Neutra lize th e pH using t he a ceta te solution. You should observe th e forma tion of a

precipita te. Cent rifuge the sa mple to pellet th e precipita te.

The rema inder of th e techn ique depends on th e exa ct pla smid prep kit being used;consult t he ma nufa cturers inst ructions for t he recommended procedure.

When t he procedure is complete, it is usua lly a good idea t o hea t t he DN A sa mple to68C for 10 minutes t o ina ct iva t e an y conta mina t ing D Nase . Af ter this s t ep, s torethe plasmid DNA a t 20C .

2 I n p re p a r in g t h e S D S /N a OH solu t ion , a d d i t i o n of t h e S D S t o N a OH a t a c on c en t r a t i o n m u c hhigher tha n t he 0 .2 M o f the f ina l so lu t ion w i l l resu l t in precip i t a t ion o f the SD S. I t i s t here forenecessary to dilute the NaOH prior to addit ion of the SD S.


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Methods: Ligation

The process of connecting two pieces of DNA together is called ligation, and isca ta lyzed by a n enzyme ca lled ligase. The liga se used m ost often in molecular biologyis derived from th e T4 bacteriopha ge, an d uses ATP to supply th e energy necessa ryfor t he reaction. In a ddit ion t o ATP , T4 l i gase requi r es DNA w i th a 5 -phospha te gr oupand f r ee 3 -hyd r oxyl gr oups.

The dra w ing a t r ight sh ow s a (very short) region ofdouble stra nded DNA. B oth stra nds of the DNAmolecule contain 5-phosphates and free 3-hydroxyl groups; this DNA molecule is thereforecapable of being ligated. This DNA fragment isblunt ended (i .e. a l l of th e ba ses ar e paired withbases f rom th e opposi te s t ra nd) ; note tha t somerestriction enzymes lea ve blunt ends, wh ile othersleave overhangs, which are short stretches of

single stra nded D NA a t either t he 5 or 3 end.

Sy nt hetic oligonucleotides cont a in free 5-hy droxylgroups, and therefore must be subjected tophosphorylation prior to ligation. In contrast,most (a lth ough not a ll) restriction enzymes leave5-phosphat e groups; most restriction fra gmentsca n be ligat ed immedia tely a fter digestion.

Ligation also requires compatible ends to the DNA. The drawings below showexa mples of different ty pes of compa tible a nd incompat ible ends (Ns imply t ha t a nya rbitr a ry sequence could be present ).

N

GTACN

5-NNNNN

3-NNNNN

AGCTN

N

5-N

3-NGTAC

N

AATTN

5-NCTAG

3-N

In compa tible 4-ba se,5-overhangs

In compa tible 4-ba se,3-overhangs

Incompatible(blun t/non-blun t ends )

NNNNN

NNNNN

5-NNNNN

3-NNNNN

CATGN

N

5-N

3-NGTAC

NN

ACNN

5-NNNTG

3-NNN

CompatibleB lunt ends

Compa tible 4-ba se,5-st icky ends

Compa tible 2-ba se,3-st icky end s

The dra w ing below shows DN A fra gments generated from restriction digests th a tused N coI a n d H indIII; these enzymes both leave 4-base 5-overhangs. The endgenerated by digestion with one of these enzymes is compatible with other endsgenerat ed by the same enzyme, but not by the ends generated by t he other enzyme.In a t t empt ing to const r uct a p la smid, scient is t s t yp ica l ly t ry to t a ke adva nta ge ofth e specif ici ty of th e restr ict ion enzymes an d of the l igase to force the creat ion apla smid w ith t he insert in t he correct orienta tion.


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In setting up a ligation reaction, it is usua lly desira ble to use a m olar excess of insertDNA relative to plasmid DNA. The insert DNA will have no effect on the cellsbeca use it lacks th e fea tur es required for replica tion. Ha ving a molar excess of insertDNA makes it more likely th a t th e ligase will find both pla smid DNA and insert D NAmolecule to connect t ogeth er, an d reduces the cha nce tha t t he incompa tible ends ofth e pla smid D NA w ill liga te to form circular pla smid D NA. (Liga tion of incompa tible

ends, a lthough very r a re, does occur; circula rized plasmid D NA will tra nsform cells,a nd w ill result in colonies tha t la ck insert.)

Ligation procedure:Mix cleav ed plas mid D NA w ith a ~ 3- to 10-fold molar excess of cleav ed insert D NA.Add ligase buffer (which includes a buffer and the ATP required to support thereaction) and ligase. Incubat e at room t emperat ure (a ctua lly, T4 liga se prefers 15 C,but for m ost rea ct ions, 20-25 C a lso a l lows eff icient l iga t ion). After 2 t o 24 hours,inactivat e the ligase by hea ting a t 68 C for 10 minutes.


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Methods: Competent Cell Preparation and Transformation

Replica tion of a plasm id requires th e insertion of the plasm id DN A into th e bacteriaused for t he replica t ion process. In genera l , bacteria a re relucta nt to ta ke up DNAfrom th eir environment (at lea st , th ey typica lly wil l not do so w ithout degrading i tf irst). In order to improve the probabil i ty t ha t t he bacteria w il l a ctua lly interna lizethe plasmid DNA, it is necessary to make the cells competent to absorb theplasm id DN A. One procedure t ha t r esults in efficient competent cells is given below .

Competent cells are signif ica nt ly m ore fragile tha n norma l bacteria . Vortexing thecells, heating the cells above 42C or to 42C for prolonged periods, or exposure of thecells to an y of a number of oth er even mildly abusive trea tm ents ma y kill th em.

Competent Cell Preparation:

TSS:85%LB medium10%PEG 80005%DMSO50 mM MgC l 2pH 6.5Ster i le f i l t er

DMSO ma y degrade in tot r a n s f o rm a t io ninhibitors; for bestresults , f resh DMSOshould be stored in frozena l i qu o t s .

G row a n overnight cultur e of the desired E. colistrain under thea ppropria te conditions (LB + a nt ibiotic, if used).

Add 1 ml a liquot of cells to 50 ml LB + 20 mM MgS O4; grow at~ 18-20 C (note: grow th a t h igher tempera tur es results insomewh a t low er efficiencies) to OD600 0.9 (i .e. mid-log phase).Cool the cells on ice for 20 minutes.Spin down th e cells (4000 rpm for 5 minu tes) in sterile t ubes.Resuspend the cells in 1/10 volume TSS (5 ml for 50 ml cultu re).

Cells ma y be used immediat ely or frozen for stora ge.Quick freeze the cells in 310 l a liquots in a dry ice:eth a nol ba tha nd st ore at -70 C.

Transformation:The procedure for allowin g competent cells to ta ke up DNA is called tr a nsforma tion.Tra nsform a tion r equires mixing a sma ll volume of DN

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472B Lab Manual 2ndEd