Creating expression vector (An overview):

1. Plasmid extraction (plasmid in which our sh-RNA is to be integrated).2. sh-RNA annealing (creating our sh-RNA/formation of oligos).3. Restriction digestion (of our extracted plasmids).4. Extracting our digested vector (Gel extraction).5. Dephosphorylation of digested vector.6. Ligation (ligating our annealed sh-RNA and dephosphorylated vector).7. Transformation (creating colonies of expressed vector).8. Lentivrus production.

a) Plasmid extraction: Alkaline lysis with SDS

• Depending on the amount of Plasmid or DNA needs to be extracted it can be classified into 3 parts:

1.Mini-preparation: requires 1-2 ml culture and yields 100ng to 5g of DNA

2.Midi-preparation: requires 10 ml culture and yields 20 g to 50g of DNA

3.Maxi-preparation: requires 500 ml culture and yields 1mg to 3mg of DNA/large scale culture.

Solutions used in Alkaline Lysis:Solution 1 (GTE solution) Solution 2 (Lysis solution) Solution 3

Glucose (50mM): acts to maintain osmotic pressure.

NaOH (0.2N): Denatures both chromosomal and plasmid DNA into single strands; the 2 strands of intact plasmid DNA remain intertwined.

Glacial acetic acid: Brings the pH back to neutral and the DNA strands can renaturate. The large chromosomal strands cannot rehybridize perfectly, but instead become partially hybridized tangle

Tris pH 8.0 (25mM): buffers the cell at pH 8.0.

SDS (1%): Dissolves the lipid component of cell membranes and cellular proteins

EDTA pH 8.0 (10mM): Binds to the divalent cations in the lipid membrane, weakening the cell envelope. Also after cell lysis EDTA limits DNA degradation by chelating Mg2+ ions that are a needed co factor for bacterial nucleases

Potassium acetate (5M): Precipitates the SDS (with lipids and proteins) from the solution. The SDS/Lipid/protein precipitate traps the tangled chromosomal DNA. This creates a “white goop” that pellets after centrifugation. Only the plasmid, small fragments of chromosomal DNA and RNA remain.

Additional solutions:

• Lysozyme solution(prepared in 1M tris pH 8.0): catalyze acid base hydrolysis of N-acetyl glucosamine and N-acetyl muramic acid residues in the proteoglycan of bacterial cell wall.• Isopropanol: precipitates nucleic acids; if allowed to sit longer will precipitate

proteins too.• Ethanol (100% and 70%): helps remove salts and any remaining SDS.• T.E. Buffer:1.Tris pH 8.0 (10mM): Buffers the DNA solution.2.EDTA pH8.0 (1mM): Binds to divalent Mg2+ ions that are a needed co factor

for bacterial nucleases limiting DNA degradation.

Generalized protocol for Plasmid extraction:

• Spin culture and obtain the cell culture pellet• Resuspend the culture in solution 1 and dissolve the pellet completely and

incubate at RT for 5 min (addition of lysozyme solution after solution 1 when doing maxi prep).• Add solution 2 mix gently and incubate at RT for 10 min.• Add solution 3 mix by shaking and incubate on ice for 10 min.• Spin at 5000rpm, 10min, 4C and collect the supernatant• Add isopropanol to precipitate the DNA at RT for 10 min. • Spin at 8000rpm 15 min at RT and dispense the supernatant.• Dissolve the pellet in 100% and 70% ethanol and give washes consecutively.• Dissolve the pellet in T.E. buffer and store at -20 C

CsCl-EtBr purification of Plasmid:

• Why Cesium chloride ??• Cesium atoms are so heavy that concentrated solution of CsCl form density gradients

after only a few hours of high speed centrifugation.• The density of initial solution of CsCl in the centrifuge tube is usually adjusted so that it

corresponds to the density of the molecule under investigation.• Densities of particles in CsCl-EtBr gradients Protein: 1.3g/ccLinear and open circular: 1.55g/ccSuper coiled:1.59g/ccRNA: >1.8g/cc

Role of EtBr:• EtBr is a 4 ring coplanar molecule that has

the ability to intercalate between the base pair perpendicular to the helical axis by making Vander Waals contact with the base pairs above and below• On an average 1EtBr molecule

intercalated / 2.5 bp causing a approximate 27% increase in the length of ds-DNA saturated with EtBr.• Reduction in the binding affinity of EtBr to

the supercoiled closed circular DNA is the basis of separation in CsCl density gradients.

CsCl-EtBr separation:

• After 70% ethanol wash, dissolve the pellet in CsCl-EtBr solution made in T.E. Buffer.• Mix well and centrifuge at 8000rpm R.T. for 10 min• Collect the supernatant and load in Ultracentrifuge tubes and spin at

60000rpm for 20hrs at 20°C• Remove the band of ccc-DNA and wash it with n-butanol so as to remove the

EtBr. Add 100% and 70% ethanol washes to remove traces of CsCl.• Discard the supernatant and dissolve the pellet in T.E. buffer and check the

purity and quantity of DNA on nanodrop.• Measure 260/280, 260/230 ratio to check the purity.

Agarose gel electrophoresis for analysis of nucleic acids:

• Principle: Separation based on the molecular size of the DNA and the type of DNA.• Gels to be cast depending upon the

product size i.e. 0.3% for 5-6 kb and 2% for 0.1 to 2 kb.• Cast gels in 1X TAE or 1X TBE buffers

with EtBr.• Mix the samples with tracking dye i.e.

bromophenol blue.• Run the Gel in electrophoresis buffer

which is either 1XTAE or 1XTBE and at 100V

b) Creating our sh-RNA oligonucleotides:

A) sh-RNA annealing:• Primers forward (100µM): 1µL

reverse (100µM): 1µL• 5X kinase buffer: 5µL• Autoclaved water: 43µL95°C for 4min70°C for 10min70°C water bath and turn it off

(overnight)

B) Kinase treatment:•Annealed product: 2µL•T4 Polynucleotide kinase: 1µL•10mM ATP: 1µL•PNK buffer: 1µL•Autoclaved water: 5µL37°C for 1hrHeat inactivation at 70°C for 10 minKeep it on ice

c) Restriction digestion of Vector:• Plasmid DNA : 2µL (extracted from maxi prep)• 10X buffer : 2µL• Age1 : 0.5µL• EcoR1 : 0.5µL• Autoclaved water : 15µLIncubation overnight at 37°C water

bathThermal inactivation 65°C for 20 minAfter digestion run on agarose gel to

separate the digested products.

e) Dephosphorylation of digested vector:

• Prevention of vector self ligation.

• Done with:a)SAP (Shrimp alkaline

phosphatase)b)CIP (Calf intestinal phosphatase)

f) Ligation reaction:

• Joining of the annealed sh-RNA and the dephosphorylated vector.• DNA• sh-RNA• T4-DNA Ligase buffer (10X).• T4 DNA Ligase • Incubation at R.T. for 2 hrs and

heat inactivation at 65°C for 10 min.

g) Transformation:

• Competent cells DH5α (EColi) and Ligated DNA mixed gently.• Heat shocked for 90s at 42°C• Added to 1ml LB and kept it on shaker at 37°C; 200rpm for 30-40 min.• Centrifuged so as to collect the pellet of transformed cells.• 100 µL LB added and the pellet was dissolved in it.• Plated on LB agar plate with Ampicillin and incubated at 37°C for 12-

16hrs for transformed colonies to appear.

RNA Extraction:

• TRIzol method (Acid Guanidinium thiocynate-Phenol-Chloroform Extraction):• Principle: A liquid-liquid extraction

technique relying on phase separation by centrifugation, into aqueous phase containing nucleic acids and organic phase containing Proteins.• Guanidinium Thiocynate: A chaotropic

agent used for denaturation of proteins, responsible for strongly binding to nucleic acid / those that are responsible for RNA degradation.

RNA Extraction: The protocol• Remove the aqueous phase and add

equal amounts of isopropanol for the precipitation of RNA.• Centrifuge for the pelleting of extracted

RNA. (Translucent pellet is an indication of presence of RNA, while Whitish pellet an indication of DNA content.)• Subsequent washing of RNA with 75 %

ethanol in DEPC water. Finally Dissolving the pellet in DEPC water.• Checking the purity and integrity of RNA

extracted.

RNA purity and Integrity:

• RNA purity: Check on Nanodrop for concentration, 260/280 ratio and 260/230 ratio.• RNA Integrity: Run RNA on 1.2% Agarose

gel prepared in 1X PBS.• Intact total RNA will have sharp clear 28S

and 18S rRNA bands (Eukaryotic). Ratio of intensity of 28S:18S rRNA should be approximately 2:1 which is a good indication of RNA being completely intact.

C-DNA synthesis:

• Reaction mixture:1.Template RNA Denature at 65°C for 5min.2.Primer (Random Hexamer) Then place on ice Make upto 20 3.DEPC water µL 25 °C (5 min)• Master mix: 42 °C (60 min)1.5X Reaction buffer 70 °C (5 min)2.Riboblock Rnase inhibitor Dispense into tubes 4 °C (∞)3.10mM dNTP mix Store DNA at -20°C

4.Revertaid M reverse Transcriptase

RT PCR:• Taq buffer: Buffers the activity of Taq

polymerase.• dNTP (10mM): Synthesis of ds-DNA.• C-DNA: provides the template for

polymerization.• Forward and Reverse primers: for the

attachment to the template strands and creating an initiation point for the reaction.

• Taq polymerase: performs the polymerization reaction and synthesizes copies of ds-DNA.

• MgCl2 (25mM): Cofactor for DNA polymerase

Run on agarose gel (2% for 200 kb and 1.2% for 1000 kb) to check for the synthesized DNA.

Protein Estimation: Modified Lowry’s method

• Principle: Under alkaline conditions the divalent copper ion forms a complex with peptide bonds in which it is reduced to a monovalent ion. Monovalent copper ion and the radical groups of tyrosine and tryptophan react with Folin reagent to produce an unstable product that becomes reduced to molybdenum/tungsten blue.• Reagents:1.CTC reagent: provides alkaline conditions and copper ions:• 0.2% CuSO4

• 0.4% Na-K tartarate• 20% Na2CO3

• 10% SDS• 0.8N NaOH2. FC Reagent: Solution of Sodium tungstate and Sodium Molybdate in Phosphoric and

Hydrochloric acid

Protein Separation: SDS PAGE • Principle: Separation of proteins according to their molecular weight, based on their differential rates of

migration through a sieving matrix (a gel) under the influence of an applied electrical field.• Reagents:

1.Acrylamide mix: Acrylamide +N,N’-methylene bis-acrylamide (29:1) for Gel Matrix

2.Sodium Dodecyl Sulphate(10%): Protein denaturant.

3.1.5M Tris base pH8.8 (in Resolving gel)

4.1M Tris base pH6.8 (in Stacking gel)

5.TEMED: performs polymerization of Acrylamide

6.Ammonium per Sulphate (10 %): Provides free radicals to TEMED for polymerization.• Sample buffer:• SDS: sample mixed with SDS and heated causes the protein to denature and linearize as SDS coats a

uniform negative charge over the protein molecules• Β-Mercatptoethanol (BME): Breaks the disulphide bond, reducing the secondary structure to primary

linearized structure.

SDS PAGE: The discontinuous system

• Employs different ions and pH in the Gel and in the electrode reservoir.• Stacking gel: pH 6.8 causes the glycine

in the Tank buffer to migrate at a slower rate as at pH 6.8 its in zwitterionic state.• Resolving gel: pH 8.8 causes the

glycine to shift from zwitterionic state to more negatively charged molecule.• Separation now takes place now

solely on the base of size of the molecule.

Western Blotting:

• Transfer Buffer constituents:1.Tris base: Maintains conductivity and pH2.Methanol: promote binding of proteins

to membrane.3.SDS: promote elution of proteins from

gel• Membrane: polyvinylidene fluoride

(PVDF) activated in methanol• Ponceau S stain: reversible staining for

visualization of transferred protein.

Immunological reactions:• Blocking: prevents non-specific background binding of the

primary and/or secondary antibodies to the membrane.• Done with non fat milk 5% (not recommended for

phosphoproteins) or 5% BSA.• Primary antibody incubation: Antibody diluted in

Blocking material for 1hr at R.T. or overnight at 4°C

• Subsequent washes with TBST to remove excess unbound primary antibody.

• Secondary antibody incubation: Secondary antibody conjugated with HRP diluted in less concentration of blocking material (1%) for 1hr at R.T.

• Subsequent washes with TBST and TBS to remove excess unbound secondary antibody.

• Develop Blots with ECL detection kit and X-ray films.

Development of blots:

• ECL constituents:1.Hydrogen peroxide soln.: In presence of

HRP coated secondary antibody H2O2 releases nascent oxygen.

2.Luminol: nascent oxygen produced oxidizes luminol giving chemiluminescence• Chemiluminescence captured using x-ray

films by exposing them for a set of intervals of time.