BT 0502 - M.Tech r-DNA Technology lab manual

IInd Semester

Department of Biotechnology

School of Bioengineering

BT 0502 r-DNA Technology lab

List of Experiments:

1. Isolation of Bacterial genomic DNA

2. Restriction enzyme digestion of pUC18 DNA

3. Purification of digested DNA by column purification

4. Ligation of DNA fragment with cloning vector

5. Preparation of Competent cells

6. Transformation in E.coli with recombinant vector

7. Polymerase Chain Reaction

8. Southern Hybridization

9. DNA Finger printing

Date:

Experiment No. 1

Isolation of Bacterial Genomic DNA

Aim:

To isolate the genomic DNA from E .coli cells.

Principle:

In prokaryotes, Deoxyribose nucleic acid (DNA) is double stranded and circular, and is found throughout the cytoplasm. The cell membranes must be disrupted in order to release the DNA in the extraction buffer. SDS is used to disrupt the cell membrane. Nucleases apparently present on human fingertips are notorious for causing spurious degradation of nucleic acids during purification. DNA can be protected from endogenous nucleases by chelating Mg2++ ions using EDTA. Mg2++ ion is considered as a necessary cofactor for most nucleases. Proteinase enzyme is used to degrade the proteins in the disrupted cell soup. Phenol chloroform is used to denature and separate protein from DNA. Chloroform is also a protein denaturant, which stabilizes the rather unstable boundary between an aqueous phase and pure phenol layer. The denatured protein forms a layer at the interface between the aqueous and the organic phases which are removed by centrifugation. DNA released from disrupted cells is precipitated by cold absolute ethanol or isopropanol.

Materials Required:

LB Broth E. coli DH5α cells Reagents

• TE buffer(pH 8.0) • 10% SDS • Proteinase K • Phenol-chloroform mixture

• 5M Sodium Acetate(pH 5.2) • Isopropanol • 70% ethanol • Autoclaved Distilled Water

Eppendorf tubes 2 ml Micropipette Microtips Microfuge

Preparation of Reagents:

1. TE Buffer (pH 8.0): 10 mm Tris HCl (pH 8.0) 1 mm EDTA (pH 8.0) 2. 10% SDS: Dissolve 10 g of SDS in autoclaved distilled water. 3. Proteinase K: Dissolve 10 mg of Proteinase K in 1 ml autoclaved distilled water. 4. Phenol-Chloroform Mixture: Mix equal volume of phenol with chloroform. Keep the mixture on ice and add 20 ml TE buffer, extract by shaking for 15 minutes. Remove the dust on the surface layer using a pipette. Repeat 4-5 times. Add 30-40 ml of TE buffer and store it on ice. 5. 5M Sodium acetate: Dissolve 41 g of sodium acetate in 100 ml distilled water and adjust pH with dilute acetic acid. 6. Isopropanol 7. 70% Ethanol

PROCEDURE:

2 ml overnight culture was taken and the cells were harvested by centrifugation for 10 minutes

875 µl of TE buffer was added to the cell pellet and the cells were dissolved in the buffer by gentle mixing.

100 µl of 10% SDS and 5µl of Proteinase K were added to the cells. The above mixture was mixed well and incubated at 37º C for an hour in an incubator.

1ml of phenol-chloroform mixture was added to the contents, mixed well by inverting and were incubated at room temperature for 5 minutes.

The contents were centrifuged at 10000 rpm for 10 minutes at 4ºC. The highly viscous jelly like supernatant was collected using cut tips and was transferred to a fresh tube.

The process was repeated once again with phenol-chloroform mixture and the supernatant was collected in a fresh tube.

100 µl of 5M sodium acetate was added to the contents and was mixed gently.

2 ml of isopropanol was added and mixed gently by inversion till white strands of DNA precipitated out.

The contents were centrifuged at 5000 rpm for 10 minutes. The supernatant was removed and 1ml 70% ethanol was added. The above contents were centrifuged at 5000 rpm for 10 minutes. After air drying for 5 minutes 200 µl of TE buffer or distilled water was added.

10 µl of DNA sample was taken and was diluted to 2 ml with distilled water.

The concentration of DNA was determined using a spectrophotometer at 260/280 nm.

The remaining samples were stored for further experiments.

PRECAUTIONS:

Cut tips should be used so that the DNA is not subjected to mechanical disruption.

Depending on the source of DNA the incubation period of Proteinase K should extended.

The phenol chloroform extraction should be repeated depending on the source of DNA to obtain pure DNA.

DNase free plastic wares and reagents should be used.

Observation:

Interpretation:

Date:

Experiment No. 2

Restriction Enzyme Digestion of pUC18 DNA

Aim:

To digest the pUC18 DNA with BamH1 enzyme

Principle:

Restriction endonucleases are the class of enzymes that are used to cleave DNA at specific sites called Restriction sites. Every restriction enzyme has a specific restriction site at which it cuts a DNA molecule. For example restriction sequence for BamHI is GGATCC. The most abundantly used restriction enzymes are type II restriction enzyme which cleaves at specific restriction site only. These endonucleases function adequately at pH 7.4 but different enzymes vary in their requirements for ionic strength usually provided by sodium chloride (NaCl) and magnesium (Mg²+) concentration. It is also advisable to add a reducing agent such as dithiothreitol (DTT) which stabilizes the enzyme and prevents its inactivation. Any variation in the concentration of NaCl or Mg can lead to changes in specificity of enzyme so that it can cleave at additional or non-standard restriction sequences.

The restriction endonucleases produce either sticky (specific e.g. EcoRI) or blunt ends (non-specific e.g. AluI) upon cleavage. Also based on the number of sequences identified for cleavage they can be tetracutter (4), hexacutter (6) or octacutter (8).

Materials Required:

pUC18 DNA

BamH1 enzyme

10X restriction buffer

1Kb Ladder

Sterile water

Agarose

6X loading dye

1.5 ml Sterile Vials

Ethidium Bromide

1X TAE buffer

Procedure:

20 µl (2.5 μg) of PUC18 DNA was taken in a fresh eppendorf. To this 23 µl of sterile water was added followed by the addition of 5 µl of 10X restriction buffer.

Finally 2μl of BamH1 enzyme was added and the mixture was incubated at 37˚C for 2-3 hrs.

0.8% agarose gel was prepared upon which 1 Kb ladder DNA, undigested pUC18 DNA and digested PUC18 DNA were run.

The gel was run at 100 V for 1 hr. The results were visualized and compared in UV illuminator.

Reaction Protocol:

PUC18 DNA : 20 µl (2.5 ug)

Sterile water : 23 µl

10X restriction

buffer : 5 µl

BamH1 : 2 µl (2 units)

-----------

Total : 50 µl

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Incubate at 37˚ C for 2-3 hours.

Observation:

Interpretation:

Date:

EXPERIMENT NO. 3

Purification of digested DNA by Column Purification

Aim:

To extract the restriction enzyme digested pUC18 DNA from the agarose gel.

Principle:

DNA elution is necessary to purify fragments of DNA or amplified PCR products from agarose gel for some application purposes like gene cloning, sequencing, restriction digestion, etc., there are so many methods by which we can elute the DNA product from the agarose gel. The following silica method is one amongst the easier methods. This isolation method of DNA is faster to perform and easier than other organic based extraction methods. It works with a wide range of sizes of DNA fragments and allows efficient recovery of the product from the agarose gel.

Guanidine thiocyanate present in the binding solution can solubilize the agarose gel piece containing the DNA fragment at 45˚C. In the presence of high salt, DNA binds silica particles present in the binding solution. Then the silica with adsorbed DNA is washed with ethanol to remove salts and other impurities from the original sample, and the clean DNA is eluted in the TE buffer. Proteins and other impurities are eliminated during washes and for this reason it is also an ideal tool to purify and concentrate DNA directly from various reaction mixtures.

Materials Required:

Silica column

Sodium iodide (6M) – 100 ml

Wash buffer – 100 ml

Isopropanol

Collection tubes

Sterile water

1.5 ml Vials

Blade\ Scalpel

Procedure:

1. Cut the DNA band of interest from the agarose gel using a sharp blade

and collect it in a micro centrifuge tube.

2. Weigh the gel and add 2.5 volumes of sodium iodide solution to it.

3. To solubilise the gel, incubate solution at 55°C for 2-3 minutes, mix

contents and further incubate for 5 minutes. The agarose gel pieces

should be completely dissolved.

4. Place the silica column on top of the collection tube.

5. Pour the contents to the silica coated column and centrifuge at

12,000rpm for 1 minute and discard the flow through.

6. Add 200 µl of wash buffer, and centrifuge at 12000 rpm for 1 minute,

and discard the supernatant.

7. Repeat steps 5 & 6 twice. Incubate the tube at 55°C for 10 minutes to

remove traces of Wash buffer thoroughly.

8. For elution of DNA, resuspend the pellet in 30 – 40 µl of water or 1X TE

buffer and incubate at 55°C for 5 minutes.

9. Centrifuge at 12000 rpm for 1 minute and collect the supernatant in a

fresh tube. Repeat the elution step once more to recover more DNA from

the silica.

10. Centrifuge once more to remove all traces of silica.

11. Check the efficiency of the elution by running the recovered on agarose

gel.

Observation:

Interpretation:

Date:

Experiment No. 4

Ligation of DNA fragments with cloning vector

Aim:

To perform the ligation of linearized vector with DNA fragment using T4 DNA ligase.

Principle:

The enzyme that joins the DNA fragments is called DNA ligases. The DNA ligase seals the nicks in DNA by formation of phosphodiester bond between adjacent 3’ hydroxyl and 5’ phosphate termini. The enzyme extensively used in joining DNA fragments is T4 DNA ligase. The Ligase joins both cohesive end as well as blunt ended DNA. The DNA fragment has poly ‘A’ at 3’ end so that it can complementally bind to the ‘T’ at the 5’ end of the vector.

Materials Required:

Restriction digested pUC18 vector and DNA fragment T4 DNA ligase Ligation buffer Nuclease free distilled water (autoclaved) Agarose Gel loading dye Ethidium Bromide Micropipettes Micro tips Microfuge 50x TAE buffer Electrophoresis unit and power supply Microwave oven/heater UV transilluminator

Procedure: Three separate vials were taken and were labelled as reaction, +ve control and –ve control.

0.5 µl (10 ng) of PCR product of DNA fragment was added to reaction and –ve control vials only.

5 µl of 2X Ligation buffer was added to each of the three vials.

1 µl (50 ng) of T- vector was added to all the three vials respectively.

1 μl of T4 DNA Ligase (1 U) was added to reaction and +ve control vials only.

2.5 μl, 3 μl, 3.5 μl of water was added to reaction, +ve control and –ve control vials respectively.

The total volume in each of the vials was 10 μl. The prepared mixtures were used for transformation in bacterial cells.

Materials Reaction +ve control -ve control

PCR product or eluted DNA fragments

0.5μl

(10 ng)

_ 0.5μl

10X Ligation buffer

5μl 5μl 5μl

pUC18 vector 1μl (50 ng) 1μl (50 ng) 1μl (50 ng)

T4 DNA Ligase 1μl 1μl _

Water 2.5μl 3μl 3.5μl

Total 10μl 10μl 10μl

Observation:

Interpretation:

Date:

EXPERIMENT NO. 5

Preparation of Competent Cells

Aim:

To prepare bacterial competent cells for transformation.

Principle:

Competence can be artificially induced in cells by treating the cells with calcium chloride prior to adding DNA. The calcium destabilizes the membrane and adheres to the cell surface favoring the formation of pores for entry of DNA. The DNA is taken during the heat shock step when the cells are exposed briefly to a temperature of 42 ˚C. Immediate chilling on ice ensures closure of pores.

Selection for cells contaminating transformed DNA is enhanced by selection markers carried by DNA. pUC series and pBR322 have ampicillin resistant factor which enables only the transformed cells to grow on Luria Bertaini-ampicillin plates.

The pUC plasmid also has the genes for β-galactosidase enzyme. The lacZ is a gene that has series of unique restriction sites engineered into it such that the plasmid be cut within lacZ gene. If the plasmids are transferred into lacZ negative strain of E. coli, they will make them lacZ positive. If either host or plasmid encoded fragments are themselves active, they can associate to form an enzymatically active protein. This type of complementation is known as α-complementation.

The lacZ positive bacteria, result from α-complementation and can produce active β-galactosidase enzyme which hydrolyses X-gal (5-bromo,4-chloro,3-indolyl β-D-galactoside) into blue colored compound and thereby appear as blue colored colonies in presence of X-gal and IPTG. Any plasmid which has been inserted with the DNA fragment in lacZ gene will

not have a functional lacZ gene and thus will produce white colonies which are unable to cleave X-gal.

Materials Required

• E.coli DH5α cells • 10mM CaCl2

• Glycerol 50%

• LB media

1g Tryptone 0.5 g Yeast extract 0.5 g NaCl Adjust to pH 7 with NaOH

• Ice

Procedure:

1. 5 ml overnight culture of E.coli (DH5α strain) cells was grown in LB media. In the morning, this culture was put back into 50 ml of fresh LB media in a 250 ml conical flask. It was aimed at diluting this overnight culture by at least 1/100.

2. The diluted culture was grown to an OD600 of 0.2 - 0.5. (a very small pellet when cells were grown 25ml to OD600 0.2).

3. 2 ml of the broth was taken in an eppendrof tube and centrifuged at 6,000rpm for 10min at 4oC.

4. The supernatant were discarded and the pellet was suspended in 1ml of 10mM CaCl2. The tubes were kept in ice for 20min.

5. It was centrifuged at 6,000rpm for 10min at 4oC. 6. Supernatant was discarded and the pellet was gently suspended in

100μl of 10mM CaCl2 and 16μl of 40% glycerol. 7. The competent cells prepared were stored at 4oC.

All subsequent steps should be carried out at 4oC and the cells should be kept on ice wherever possible

Observation:

Interpretation:

Date:

Experiment No. 6

Transformation in E.coli with recombinant vector

Aim:

To transfer recombinant vector into competent bacterial cells.

Principle:

Transformation mainly involves the uptake up foreign DNA into the cell from the medium. Selection for cells containing transformed DNA is enhanced by selection markers carried by DNA. pUC series and pBR322 have ampicilllin resistant factor which enables only transformed cells to grow on Luria Bertaini – ampicillin plates.

The pUC plasmid also has genes for β-galactosidase enzyme. The lacZ is a gene that has series of unique restriction site engineered into it such that the plasmid is cut with in lacZ gene. If the plasmids are transferred into lacZ negative strain of E.coli, they will make them lacZ positive. If either the host or the plasmid encoded fragments are themselves active, they can associate to form an enzymatically active protein. This type of complementation is known as α-complementation.

The lacZ positive bacteria, result from α-complementation and can produce active β-galactosidase enzyme which hydrolyses X-gal (5-bromo, 4-chloro, 3-indolyl β-D-galactoside) into blue colored compound and thereby appear as blue colored colonies in presence of X-gal and IPTG. Any plasmid which has been inserted with the DNA fragment in lacZ gene will not have a functional lacZ gene and thus will produce white colonies which are unable to cleave X-gal.

Materials Required:

• Balance • pH meter • Microcentrifuge • Autoclave/Microoven, • Incubator • Shaker, Refrigerator

Chemicals/Reagents:

• LB Broth • Agar-Agar • Ampicillin (100μg/ml) • X-gal stock: 20mg is dissolved 1ml of dimethyl

formamide. Store at -20oC. • IPTG Stock: 20mg of IPTG was dissolved in 1ml of sterile

water. • IPTG/X-gal: Add 800μl of IPTG stock to 3.8ml of sterile

water. Swirl well to mix. Add 400μl of X-gal stock and mix. The solution should be colorless. Use 250μl per plate. The solution can be stored at -20oC.

Procedure:

Preparation of LB (Luria Broth) plates:

1. Bacto-Tryptone (2.5g) + Bacto-Yeast Extract (1.25g) + NaCl (2.5g) adjust pH (7–7.5).

2. Add 200ml distilled H2O and stir until it dissolves and make up the volume to 250 ml

3. Autoclave or keep the flask in micro-oven (to melt the agar).

4. Take out the flask & cool to 45°C - 50°C.

5. Add 250μl Ampicillin (50 mg/ml) (final concentration: 50 μg/ml).

6. Without making air bubble gently swirls the solution and pour the solution in bacterial petridishes & let them solidify.

Concentrations of commonly used antibiotics:

Antibiotic Stock solutions

Concentration Storage Working concentration

(dilution)

Ampicillin (sodium salt)

50 mg/ml in water - 20°C 50-100 μg/ml (1/500 – 1/1000 )

Chloramphenicol 34 mg/ml in ethanol -20°C 170 μg/ml (1/200)

Kanamycin 10 mg/ml in water -20°C 50 μg/ml (1/200)

Streptomycin 10 mg/ml in water -20°C 50 μg/ml (1/200)

Tetracycline HCl 5 mg/ml in ethanol -20°C 50 μg/ml (1/100)

Carbenicillin 50 mg/ml in water -20°C 50 μg/ml (1/1000)

Transformation:

1. Mix 1–2 μl of DNA (1-10 ng) with 100 μl E. coli competent cells (DH5α) in pre-chilled tubes.

2. Swirl on ice for every 5 min & incubate on ice for totally 30 min

3. Incubate tubes in water bath at 42°C for 45 sec & incubate on ice for 5 min

4. Add 600 μl of LB and put in a bacterial shaker for 1hr at 37°C.

5. Plate 5, 10, 20, 50 & 100 μl of culture onto LB plates containing ampicillin (100μg/ml) with IPTG and X-gal (conc 200μg/ml).

6. Keep them in a bacterial incubator (37°C) for over night.

Observation:

Interpretation:

Date:

Experiment No. 7

Polymerase Chain Reaction

Aim: To perform PCR amplification of specific target sequence from genomic DNA and to analyze the amplified product by agarose gel electrophoresis.

Principle:

The polymerase chain reaction (PCR) is an in-vitro DNA amplification of target DNA with a pair of primers and a DNA polymerase, resulting in several million fold amplification of the target sequence within few hours. In this way, the sample (target DNA) is allowed to react with a pair of primers (specific to each target sequence), deoxynucleotide triphosphates, buffer and taq DNA polymerase. Of the primers are complementary DNA a new DNA strand are synthesized. During each cycle, the DNA strand is doubled. Each cycle consists of 3 segments viz., a denaturation step, during which the 2 strands of target DNA is separated ; an annealing step, during which the primers attaches to the complementary target sequences and a synthesis step during which a new strand is synthesized with the help of dNTPs and enzyme.This technique has been used for specific detection of various bacterial and viral pathogens from clinical samples.

Materials Required:

1. Primers: Forward and Reverse

2. dNTPs: dATP, dGTP, dCTP, dTTP.

3. Taq DNA polymerase.

4. MgCl2

5. PCR Buffer (10X)

6. Template DNA: genomic DNA

Procedure:

1. Setting up PCR:

Add the following reagents to the PCR tube in the following order

Sterile water 38ul

10X assay buffer 5ul

10mM dNTP mix 3ul

Template DNA 1ul

Forward primer 1ul

Reverse primer 1ul

Taq DNA polymerase 1ul

Total reaction volume 50ul

2. Mix the contents gently and layer the reaction mix with 50ul of mineral oil to evaporation.

Note :- mineral oil need not be added if the thermocycler is equipped with a heated lid.

3. PCR amplification:

Carry out the amplification in a thermocycler for 30 cycles using the following reaction conditions;

a. Initial denaturation - 940C 1minute

b. denaturation - 940C for 30 seconds

c. annealing – 480C for 30 seconds 30 cycles

d. extension- 720C for 1 minutes

e. Final extension - 720C for 2 minutes

4. Analysis on Agarose gel:

a. Following PCR amplification, add 5ul of gel loading buffer to each of the PCR tubes.

b. Tap the mixture thoroughly and wait for a few seconds for the 2 layers to separate.

c. Carefully pipette out 15ul of reaction mixture (avoiding mineral oil layer) and load onto 1.5% agarose gel.

d. load 10ul of the ready to use marker provided. Note down the order in which the samples have been loaded

e. Run the samples at 100V for 1-2 hours till the tracking dye reaches 3/4th of the length of the gel.

f. Visualize the gel under UV transilluminator.

Observation:

Interpretation:

Date:

Experiment No. 8

Southern Hybridization

AIM:

To learn the technique of southern hybridization involving the following experiments:

1. Electrophoretic separation of DNA by agarose gel electrophoresis

2. Electrophoretic transfer of DNA from agarose gel to Nylon membrane

3. Immobilization of DNA onto nylon membrane

4. Hybridization and non-isotopic detection of DNA of interest.

PRINCIPLE:

Southern hybridization technique involves transfer of DNA fragments separated in electrophoretic gels to membrane filters for detection of specific base sequences by complementary probes. Prof. E.M. Southern developed this technique in 1975, hence referred to as Southern transfer. Southern blots are uised to identify and quantitate specific DNA sequences, in analysis of genome organization and expression, in the study of genetic diseases, in DNA fingerprinting and analysis of PCR products.

In this technique, DNA molecules are size fractionated on a gel and transferred to a nitrocellulose or nylon membrane by capillary or electrophoretic transfer. The DNA is immobilized onto the membrane by UV crosslinking or by baking at 80O C. The membrane is washed, prehybridized and then hybridized with a biotin labeled probe. After hybridization, the unbound probe is remove by washes. The membrane is then incubated with protein block to reduce non-specific interaction. The bound (hybridized) probe is detected by incubating the membrane with streptavidin enzyme conjugate and finally incubated with the substrate solution until sufficient color (blue) develops.

MATERIALS REQUIRED:

1. 25N Hydrochloric acid (1liter)

2. Denaturation Solution (1liter)- stored at RT

87.75g Sodium chloride (1.5M)

20.09g Sodium hydroxide (0.5M)

PROCEDURE:

Day1: AGAROSE GEL ELECTROPHORESIS:

1. Prepare a 1% agarose gel containing ethidium bromide.

2. Load 20ul of ready to use DNA marker supplied onto the gel. Run at 50-100V until the dye reaches 4.5cm from the well.

3. Cut the DNA marker lane from the agarose gel as follows:

Cut the gel ~3mm above the first band and ~2mm below the last band, ensuring the gel measures about 4 to 4.5cm.

Note: Mark the appropriate position of the gel to be cut on the gel tank. Take care not to expose yourself to UV light.

4. Cut the filter paper (2nos) and nylon membrane exactly to the size of the cut gel. Ensure there is no protrusion of the filter paper and the membrane from the gel.

5. Wet the cut gel, Nylon Membrane, Filter Papers and the electrotransfer cassette in 1X Electrotransfer buffer.

Electroblotting:

Assemble the electrotransfer apparatus as shown below. Start the arrangement by placing filter paper on the cathode cassette cover following by the cut gel and Nylon Membrane. Mark and place the soft side of the Nylon Membrane to the cut gel. Place the wet filter paper on the Nylon Membrane followed by anode cassette cover. Tighten the electrotransfer cassette tightly with the screws provided.

NOTE: Ensure no air bubbles are present between any of the layers of filter paper, cut gel and the Nylon Membrane.

6. Insert the cassette into the apparatus filled with 250 ml of 1X Electrotransfer Buffer.

7. Connect the cords to power supply according to the convention red:anode, black:cathode and set voltage to 50 V for 3 1/2 hours.

8. Turn off the power supply and remove the cassette from the apparatus. Drain the buffer.

Immobilization of DNA on membrane:

9. Remove the Nylon Membrane gently from the cassette and place it on a thin transparent polythene sheet and place this on a UV transilluminator (expose the soft side of membrane containing transferred DNA to UV light) with UV lamps switched on for 20 minutes. This helps in fixing the DNA on the membrane.

NOTE: Do not expose yourself to UV light.

10. Turn off the UV transilluminator. Place the membrane in plastic petridish provided and incubate in a hot air oven at 70oC for 30 minutes. This ensures complete immobilization of DNA onto the membrane.

Hybridization:

11. Bring the petridish containing the membrane to room temperature after incubation. Add 10 ml of Prehybridization buffer to the petridish and incubate at 45oC incubator shaker with mild shaking (70-90 rpm) for 45 mins.

12. After incubation, discard prehybridization buffer taking care not to discard the membrane.

13. Add 10 ml of Hybridization buffer to the petridish containing the membrane.

14. Keep 1 vial of Biotinylated probe for 10 minutes in a boiling water bath and immediately chilli by placing it on ice for 5-10 minutes.Add this probe to the Hybridization Buffer in the Petridish. (Rinse the probe vial with 300μl of hybridization buffer and add it to the petridish). Incubate the petidish at 45oC incubator shaker with mild shaking 70 rpm for 16 hours.

Day 2: Blocking and detection

15. Decant the hybridization buffer,add 10 ml of 1X wash buffer A and gently swirl the petridish for 5 mins at room temperature. Reapeat the washes twice (each wash of 5 mins)

Note: discard the buffer after each wash.

16. Add 10 ml of 1X prewarmed(70oC) wash buffer B and gently swirl the petridish. Incubate at 70oC for 5 mins in a hot air oven and gently swirl. Repeat the washes for another two times.

Note: Discard the buffer after each wash.

17. Add 10ml of 1X blocking buffer to the petridish and incubate at room temperature for 1 hr with gentle rocking.

Note: Discard the blocking buffer.

18. Add 9ml of diluted HRP-Streptavidin conjugate to the petridish and incubate at room temperature for 20min with gentle rocking.

Note: Discard the conjugate buffer.

19. Add 10 ml of 1X wash buffer C to the petridish and incubate at room temperature for 5 minutes with gentle rocking. Repeat the washes two more times. Note: Discard the buffer after each wash.

20. Add 10 ml of 1X wash buffer D to the petridish and incubate at room temperature for 5 minutes with gentle rocking. Repeat the washes two more times. Note: Discard the buffer after each wash.

21. Add 5 ml of 1X substrate solution and gently swirl at room temperature for 20-45 minutes until a blue colour band develops.

22. After blue colour band is seen, stop the reaction by placing the membrane in distilled water.

Observation:

Interpretation:

Date:

Experiment No. 9

DNA Fingerprinting using RAPD Technique

Aim:

To perform DNA fingerprinting by random amplification of polymorphic DNA (RAPD) technique by PCR.

Principle:

RAPD (Random Amplified Polymorphic DNA) is a PCR based technique that makes use of random primers that bind to a number of partially or perfectly complimentary sequences at unknown locations in the genome of an organism and produce specific bands (DNA fingerprints) that are unique. Due to variations in genomic DNA, the number and sizes of the amplified product will vary exhibiting genomic polymorphism. Random Amplified Polymorphic DNA (RAPD) is a general technique employed for obtaining the molecular fingerprint of a strain or species.

Materials Required:

• Taq DNA Polymerase • 10X Assay Buffer • Random Primer • 10mM dNTP Mix • Mineral oil • Test genomic DNA • Serratia marcescens genomic DNA • Bacillus subtilis genomic DNA • E.coli B genomic DNA • E.coli k12 genomic DNA • Test genomic DNA

Procedure:

Setting up PCR:

1. Prepare a cocktail of PCR reaction mix for five PCRs with five different target DNA samples. The variable components (four control genomic DNA and one test DNA) are to be added separately.

Preparation of PCR reaction mix:

2. Add the following reagents to one PCR tube in the following order: • Sterile water - 76μl • 10X assay buffer - 10μl • 10mM dNTP mix - 7.5μl • Random primer (100ng/μl)- 6.25μl • Taq DNA polymerase - 2.5μl

Mix the contents gently. All the additions should be done on ice.

3. Aliquot 20μl of the above reaction mix to each of the five different PCR vials placed on ice and label as 1,2,3,4 and 5.

4. Add 1 μl of Serratia marcescens genomic DNA to vial labeled 1. Similarly add 1 μl each of genomic DNAs of Bacillus subtilis, E.coli B,

E.coli k12, test DNA sample to the vials labeled 2, 3, 4 and 5 respectively.

5. Mix the contents gently and overlay with 50 μl of mineral oil to prevent evaporation.

6. Centrifuge the samples briefly (6000rpm for 30 sec at 4oC) to bring down the contents of the tube.

PCR amplification:

7. Carry out the amplification using a thermocycler for 45 cycles according to the following condition:

a. Initial denaturation - 950C 3mins

b. Denaturation - 950C for 1 min

c. Annealing – 450C for 1 min 45 cycles

d. Extension- 720C for 2 mins

e. Final extension - 720C for 5 mins

8. Following PCR amplification, add 5 μl of gel loading buffer to each of the PCR tubes.

9. Perform agarose gel electrophoresis with 2% gel and load the samples. Run at 100V for 1 hour.

Observation:

Interpretation: