Team:VIT Vellore/Documentation

(Difference between revisions)

Amplification:

Reaction Mixture-
- 1µl of samµple DNA template
- 2µl of 10X buffer
- 1.2µl of MgCl2
- 2µl of dNTPs
- 0.5µl of KOD DNA polymerase
- 2.0µl of forward and reverse primers each
- 9.3µl of water to bring reaction volume to 20ul
Thermocycler-
- 94*C for 5 mins, initialisation
- 30 cycles of:
- 94˚C for 1 min, Denaturation
- 55˚C for 1 min, Annealing
- 72˚C for 3min, Elongation
- 72˚C for 10 mins, Final Elongation
- 4˚C indefinitely, Final Hold

Agarose Gel Electrophoresis
- The agarose gel is prepared with BromoPhenol Blue added as tracking dye.
- The lengths of each part are noted down.
- 20ul of the samples and 7ul of an appropriate ladder are loaded and kept for electrophoresis at 100V for 45 mins.
- After running, the gel is taken to the gel doc machine, and the image under UV transillumination is captured.

Restriction Digest

Prepare the reaction mixture at room temperature in the order indicated:

Component Volume Water, nuclease-free 15 µl 10X FastDigest® buffer 2 µl DNA 2 µl (up to 1 µg) FastDigest® enzyme 1 µl Total volume 20 µl

Mix gently and spin down

Incubate at 37°C in a heat block or water thermostat for 5 min.

Gel elution

Materials:
1. Agarose Gel
2. Tris-EDTA (T.E)
•  10mM Tris-HCl pH 7.6
•  1mM EDTA
3. Isopropanol/ phenol
4. Chloroform
5. Ethanol
6. Sodium acetate
Procedure:
1. Run DNA on "Low melt" agarose gel
2. After band have separated, visualize band on UV box, cut band out
3. Add 100ml of T.E to band, crush, heat to 65oC for approx. 5 min, add 200ml of phenol/ isopropanol, vortex, heat 65oC 3 min., vortex
4. Microfuge 5 mins, remove supernant
5. Add 100ml of T.E. to phenol/isopropanol, vortex, heat 65oC 3 min, vortex
6. Microfuge, pool supernants.
7. Chloroform extract (approx. 400 ul), microfuge 3 min.
8. EtOH precipitate, adding 1/10 vol. 3M Na0Ac, 2.5 vol. EtOH.
9. -20oC 1-2+hrs; spin 30 min., dry down, bring up in suitable vol 0.1X T.E.
Precautions:

1% -600 bp to 5kb; 2% 300700bp; Smaller? Use 2% "Nusieve" + 1% "Low melt".

Minimize exposure of DNA to UV, DO NOT scratch filter on box.

Ligation of Vector and Insert

Reaction Mix

3µl of Vector DNA

5µl of Insert DNA

1µl of Ligase

7µl of water

After adding the reaction mix, shake the epp. tube/ PCR tube gently
Let the mixture stand at Room Temperature (25oC) for 5 min.
The ligation is complete.
Perform an AGE to confirm the success of the reaction.

Transformation and competent cell preparation:

Chemical competent cell preparation


Materials:

1. Culture media
2. LB Broth
3. CaCl2
4. glycerol
Procedure:
1. Grow bacteria.
2. Inoculate 1:100 into LB.
3. Grow until OD @ 600 reaches 0.4-0.6.
4. Take a 2ml aliquot.
5. Centrifuge at 6000rpm for 8 min at 4˚C.
6. Resuspend the pellet in 800µl ice cold CaCl2.
7. Chill on ice for 30 min.
8. Centrifuge at 6000rpm for 8 min at 4˚C.
9. Re-suspend the pellet in70µl ice cold CaCl2 + 10µl glycerol.
9. Freeze the cells.

Transformation

1.Competent cells had been prepared previously and stored.
2.The competent E.coli cells are taken and thawed on ice.
3.Next the DNA (5µl) is added to the cells and undergo heat shock.
4.The mixture is incubated at 42oC for exactly 90 seconds and then immediately placed on ice.
5.Left for 1 hour incubation.
6.The transformation is complete.
7.The transformed cells are plated and screened using a suitable antibiotic.
8.Preparation of Antibiotics

9.Take ampicillin stock and dilute to a concentration of 100mg/ml with sterile water.
10.Mix thoroughly and using a sterile syringe, withdraw all of the solution from the vial.
11.Remove the needle of the syringe and attach a single use MiniSart filter unit.
12.Use the plunger to filter out the ampicillin solution into 1.5 ml epp. tubes
13.Plating on Ampicillin Plates

14.The transformed cells are poured onto LB Agar plates containing Ampicillin.
15.Spreading is done by introducing 2-3 glass beads into the plate and then shaking vigorously in a lateral direction.
16.The plates are incubated at 37oC for 24hrs.
Materials:

LB broth bacteria culture medium. The content are 1% Tryptone, 0.5% yeast extract, 200 mM NaCl, then it sterilize by autoclaving in suitable aliquots.
STET mix which is contain 5% (v/v) Triton X-I 00, 50 mM Tris-HCl, pH 8.0, 50 mM EDTA, pH 8.0, 8% (w/v) sucrose. Store the mix solution at room temperature.
Lysozyme: Dry powder. Store at -20oC.
70% Ethanol.
Isopropanol.
TE solution: 10 mM Tris-HCl pH 8.0,1 mM EDTA.
A boiling water bath: An opened bottom tube rack is required because the tubes must be placed directly in the water to achieve rapid heating.
Sterile wooden toothpicks.
Methods:

1. Set up a culture for each miniprep by inoculating 2-3 mL of L-broth, containing an appropriate antibiotic (e.g., 100 micrograms/mL ampicillin) with a bacterial colony. Grow overnight at 37oC with vigorous shaking.
2. Where plasmids have a high copy number, the growth time may be reduced to approx 6 h.
3. Before starting the miniprep, begin boiling the water and make up a fresh solution of 1 mg/mL lysozyme in STET mix.
4. Fill a 1.5-mL labeled microfuge tube with an aliquot from each culture. Pellet the bacteria by centrifugation for 1 min at 12,000 g. Carefully aspirate off the supernatant using a drawn-out Pasteur pipet.
5. The short centrifugation time leaves a loose pellet that is easier to resuspend. If the pellet does not readily resuspend, pipet the solution up and down to dislodge it. Do not suck the pellet directly into the pipet tip.
6. Vortex each pellet for a few seconds to break up the pellet. Add 20 micrliters STET mix to each tube. The pellet should now easily resuspend by vortexing.
7. Immediately place the tubes in the open-bottom rack, and place in the boiling water for exactly 45s. Ensure that each tube is at least half submerged.
8. Centrifuge the tubes at 12,000 g for 10 min. A large, sticky, loose pellet should form.
9. Remove the pellet from each tube by “fishing” it out with a sterile wooden toothpick. Because the pellet is quite slippery, it is useful to have a paper tissue at the top of the tube to catch the pellet and prevent it from slipping back down into the tube.
10. Add 200 microliters isopropanol to each tube, and centrifuge at 12,000 g for 5 min.
11. Aspirate the supernatant, and wash the pellet in 500 microliters 70% ethanol. Centrifuge the tube for 1 min to compact the pellet, and then aspirate the 70% ethanol.
12. Air dry the pellets for 10 min, and resuspend each one in 100 microliters TE buffer. Vortex and shake for 10 min before use to ensure complete dissolution.
13. Use 10 microliters (equivalent to 100 ng of plasmid for most vectors) and analyze by gel electrophoresis.
Colony PCR


14.Use a sterile toothpick or pipette tip to re-suspend a plated colony in 50μl sterile water.
Reaction Mix

1 μl 10x polymerase buffer
1 μl 10x dNTPs (10x = 2.5 mM each dNTP)
0.15 μl 40 μM FWD primer
0.15 μl 40 μM REV primer
0.1 μl Polymerase
6.6 μl H2O
1.0 μl template suspension
PCR protocol

95 C for 6 minutes (disrupt cells, separate DNA)
Cycle 35 times:
95oC for 30 s (melting)
53oC for 30 s (annealing)
72oC for 45 s (elongation)
72oC for 10 minutes (final elongation)
4oC for storage.
Isolation of bacterial plasmid

Materials:
1. TENS solution:
* 10 mM Tris (pH to 7.5)
* 1 mM ethylenediaminetetraacetic acid (pH to 8.0 to dissolve)
* 0.1 N sodium hydroxide
* 0.5 % sodium dodecyl sulfate
2. 3 M Sodium acetate, pH 5.2
3. Pre-chilled (at -20 degrees C) 100 % ethanol
4. 70 % Ethanol
5. Distilled water
6. Overnight bacterial culture
Supplies:
1. Micropipetter and tips
2. Vortex mixer
3. Microcentrifuge and tubes
Procedures:
1. Spin 1.5 ml of overnight bacterial culture for 30-60 seconds in a microcentrifuge.
2. Decant supernatant, leaving 50-100 ul in the tube.
3. Vortex to resuspend the bacteria pellet completely.
4. Add 300 ul of TENS solution.
5. Vortex for 5 seconds to mix.
6. Add 150 ul of the sodium acetate.
7. Vortex for 5 seconds to mix.
8. Spin for 2 minutes in a microcentrifuge.
10. Add 0.9 ml of pre-chilled 100 % ethanol.
11. Spin for 5 minutes in a microcentrifuge.
12. Discard supernatant and add 1 ml of 70 % ethanol.
13. Discard the ethanol and add another 1 ml of 70 % ethanol.
14. Withdraw and discard as much liquid (ethanol) as possible then air-dry the pellet.
15. Resuspend the pellet in 30 ul of distilled water and keep at 4 degrees C or -20 degrees C.
Precautions: DNA should be stored at -20 degrees C, but repeated freeze-thaw should be avoided. So if it is needed frequently or in a couple of days, store it at 4 degrees C.