Team:VIT Vellore/Documentation

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Overall Project Flowchart - General

Protocols

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

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.

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.