Team:IIT Delhi/Protocol

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Competent Cell Preparation

1. Inoculate Escherichia coli DH5α culture in a test tube containing 5ml LB media and put on overnight incubation at 37⁰C.
2. Inoculate 1ml of the overnight culture in a 100ml flask for 2-3 hours to get an Absorbance of 0.4 to 0.6 so that cells reach the early log phase.
3. Centrifuge at 3000g for 10mins at 4⁰C.
4. Resuspend for 5 mins in 10 ml (for each oak ridge tube) 0.1M CaCl2 solution without vortexing.
5. After resuspension, keep on ice for 30mins.
6. Centrifuge at 3000g for 10mins at 4⁰C.
7. Resuspend the whole cell pellet in 2-3ml 0.1M CaCl2 /15% Glycerol solution and dispense in 100uL aliquots.

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Transformation

1. Thaw competent cells on ice. Label 2.0ml microcentrifuge tubes for each transformation and pre-chill by placing the tubes on ice.
2. Pipette the DNA (1uL in case of the Transformation Efficiency Kit and whole mixture in case of ligation product) into each microcentrifuge tube.
3. Pipette 50ul of competent cells into each tube. Flick the tube gently with your finger to mix. Incubate on ice for 30 minutes. Pre-heat waterbath now to 42°C.
4. Heat-shock the cells by placing into the waterbath for 1 minute. Be careful to keep the lids of the tubes above the water level, and keep the ice close by.
5. Immediately transfer the tubes back to ice, and incubate on ice for 5 minutes. This helps the cells recover.
6. Add 200ul of LB media per tube, and incubate at 37°C for 1-2 hours. Prepare the agar plates during this time and label them.
7. Pipette 100ul from each tube onto the appropriate plate, and spread the mixture evenly across the plate.
8. Incubate at 37°C overnight or approximately 16 hours. Position the plates so the agar side is facing up, and the lid is facing down.

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Plasmid Isolation (Qiagen MiniPrep Plasmid Isolation Kit©)

1. Inoculate the cells containing the plasmid in 5ml LB tube and incubate overnight at 37⁰C.
2. Pellet the cells at 12000rpm for 1 min.
3. Discard the supernatant, resuspend the cell pellet in 600uL MilliQ and again centrifuge at 12000rpm for 1min.
4. Discard the supernatant and add 250uL Buffer P1 to resuspend the whole pellet. Solution should become opaque.
5. Gently, add 250uL Buffer P2 (lysis buffer) and pipet two-three times to observe a clear solution. Invert the sample 4-6 times to mix the solution thoroughly. Do not keep for more than 2-3 minutes.
6. Add 350uL Buffer N3 (Neutralization) and pipet two-three times to form a cloudy mass above a clear solution (containing plasmid DNA). Invert 4-6 times to mix thoroughly.
7. Centrifuge the solution at 13000 rpm for 10 mins to completely pellet the cloudy mass.
8. Pipet out the supernatant, taking care not to disturb the pellet, and put into a spin column that comes with the kit. Label the column before proceeding.
9. Centrifuge at 12000 rpm for 1 min and discard the flow through.
10. Add 750uL PE Buffer (make sure 70% ethanol was added previously) and repeat step 9.
11. Dry centrifuge the column and repeat step 9 to remove the excess PE Buffer.
12. Take a new 1.5ml microcentrifuge tube and put the column in it. Now add 60uL EB (Elution Buffer) directly at the center of the column matrix.
13. Keep at Room Temperature for 10-15mins and centrifuge for 1min at 12000 rpm.
14. Store in the -20⁰C freezer.

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Restriction Digestion (Single Reactions)

Materials
20ul DNA
2.3uL Buffer
0.5uL Restriction Enzyme(s)

Procedure

Mix all the above materials in a microcentrifuge tube/PCR tube and incubate in a waterbath for 2 hours at 37⁰C. Run on an appropriate percentage Agarose Gel to check digestion.

1% Agarose Gel Electrophoresis

1. Weigh 0.4g agarose and put into 40ml 1X TAE Buffer in a flask.
2. Mix the contents thoroughly by heating up in a microwave and keep at Room Temperature for 10-15mins.
3. Clean up the tray and set up the chassis for Electrophoresis.
4. After sufficiently cooling the contents in the flask, add 5uL Ethidium Bromide (Diluted) and mix by shaking.
5. Pour the contents into the tray, let it solidify. Remove the comb, load the sample along with 6X bromophenol blue dye and switch on the current.
6. Observe under UV light to get the required sizes of DNA bands.

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Gel Elution (Qiagen Gel Extraction Kit©)

1. Carefully cut part of the gel containing the DNA fragment to be eluted using a clean, sharp scalpel and keep it in a 1.5ml microcentrifuge tube.
2. Weigh it and add 3 X (Volume of Gel) in uL of QG buffer. For example, if the Gel weighs 300mg, add 900uL QG Buffer.
3. Incubate the solution in a waterbath (preheated to 55⁰C) and vortex thoroughly after ever 2-3 mins. It is important that the solution turns homogeneous.
4. Add 1 X (Volume of Gel) in uL of isopropanol and mix.
5. Add the solution to the quick spin column in a provided 2 ml collection tube and label appropriately.
6. Centrifuge at 12000 rpm for 1 min and discard the flow through.
7. Add 750uL PE Buffer (make sure 70% ethanol was added previously) and repeat step 6.
8. Dry centrifuge the column and repeat step 6 to remove the excess PE Buffer.
9. Take a new 1.5ml microcentrifuge tube and put the column in it. Now add 35uL EB (Elution Buffer, preheated to 60⁰C) directly at the center of the column matrix.
10. Keep at Room Temperature for 10-15mins and centrifuge for 1min at 12000 rpm.

Store in the -20⁰C freezer.

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Ligation

Materials
MilliQ
Vector DNA
Insert DNA
T4 DNA Ligase
Ligase Buffer

Procedure

1. Mix Vector and insert in 1:3 ratio, 1uL Buffer, 0.5uL ligase enzyme and make up to 10uL using MilliQ in a microcentrifuge tube/PCR tube.
2. Incubate for 3 hours at 22⁰C.
3. Incubate for 2 hours at 16⁰C.
4. Store at 4⁰C.

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PCR Fragment Amplification

Materials
MilliQ
Taq/Deep Vent Polymerase
10X Buffer for Taq/MgSO4 for Deep Vent
dNTPs
Template DNA
Forward Primer
Reverse Primer
Template DNA
Procedure

Mix the above contents in a PCR tube and set the following program on the PCR machine
1. 95⁰C for 3mins
2. 95⁰C for 1min
3. 62⁰C for 1min (This can change depending on the Tm of the primers)
4. 72⁰C for 1min (This can change depending on the amplicon length)
5. Repeat steps 2-4 30 times
6. 72⁰C for 10mins
7. 4⁰C forever

Run on the appropriate percentage agarose gel to check the PCR product.

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Colony PCR

*Same as PCR, except that a whole cell is used as template instead of pure DNA and step 1 in the PCR program extends to 10mins at 95⁰C to completely lyse the cells.





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References

[1] A modified protocol, taken from Molecular Cloning- Sambrook & Russel- Vol. 1, 2 & 3.
[2] A modified version of the iGEM Transformation protocol .