Easy to use Protocols for Molecular Biology and Biotechnology Labs

Primer designing (principles)

1.Determination of particular restriction sites on vector as well as in the primers
2.Make it sure that same restriction site is not present within the coding region of the target gene.
3.Primers should not be complementary to each other in order to avoid primer dimmer formation.
4.Primers should not contain less than 50% GC content
5.Annealing temperature of primers should be considered.

Agarose gel electrophoresis

DNA fragments are separated by electrophoresis on 1% (w/v) agarose gels in 0.5 X TAE buffer containing ethidium bromide (10 mg/ml). Fragment sizes can be estimated by comparining with 1kb DNA ladder.

50X Tris-acetate EDTA buffer (TAE)

Tris base 242 gm

Glacial acetic acid 57.1 ml

0.5 M EDTA (pH 8.0) 100 ml

Make up the final volume with distilled water to 1000 ml.

6X Gel loading buffer

Bromophenol blue 0.25% (w/v)

Xylene cyanol FF 0.25% (w/v)

Glycerol 30.0% (v/v)

Dissolve in distilled water

LB (Luria-Bertini) medium

Tryptone 1.0 %

Yeast extract 0.5 %

NaCl 0.5 %

Agar 1.5 %

Mini prep (Plasmid Isolation) solutions

Solution I (suspension buffer)

Tris (pH 7.4-7.6) 50 mM

EDTA 1 mM

RNase 100 μg/ml

Solution II (Denaturation soln.)

NaOH 0.2 N

SDS 1 %

Solution III (Neutralization soln.)

Potassium acetate 3 M

Glacial acetic acid 11.5ml/100ml (pH 4.8-5.0)

Plasmid isolation from E.coli: alkaline lysis method (miniprep)

Following protocol can be used for the isolation of plasmid DNA from E. coli.

1. Culture a single E. coli colony in 3 ml liquid LB medium containing antibiotics (some times 100 mg/ml ampicillin or 50 μg/ml kanamycin) and grow overnight at 37 °C in an incubator shaker.

2. Centrifuge 1 ml of E. coli culture in 1.5 ml eppendorf tube at 14000 rpm for 1 minute.

3. Discard the supernatant and air dry the pellet.

4. Add 200 ml of solution I to eppendorf tube and suspend the pellet in the solution with the help of vortexer machine.

5. 200 ml of solution II was added to eppendorf tube and mixed well by inverting gently and incubate for 5 minutes at room temperature.

6. Add 200 ml of solution III to eppendorf tube, mix well and incubate for 5 minutes at room temperature then centrifuge at 14000 rpm for 5 minutes.

7. Take the supernatant in a fresh eppendorf tube and add two volume of pre-chilled 100% ethanol or Isopropanol.

8. Incubate the tubes at -20 °C for 20 minutes and then centrifuge at 14000 rpm for 10 minutes.

9. Discard the supernatant, wash the pellet with 70% ethanol and air-dry the pallet.

10. Dissolve the DNA in 20 ml of sterile distilled water was stored at -20 °C.

The plasmid concentration and quality can be detected by 1% agarose gel using stranded DNA markers.

Rapid gel extraction protocol (Quick jet Kit)

NOTE

ü Perform all centrifugations at room temperature.

ü Preheat an aliquot of TE to 65-70 ºC before starting the experiment.

ü Equilibrate a water bath or heat block to 50 ºC.

ü Verify that ethanol has been added to wash buffer (L2).

1. Gel slice excision

Cut the area of gel containing the DNA fragment using a clean, sharp blade. Minimize the amount of surrounding agarose excised with the fragment.

2. Gel slice weighing

Weigh the gel slice.

a. For < 1% agarose gel, place up to 400 mg of gel into a 1.5 ml polypropylene tube.

Divide gel slices exceeding 400 mg among additional tubes. Add 30 μl of Gel solubilization buffer (L1) for every 100 mg of gel.

b. For >1% agarose gel, place up to 400 mg of gel into a 5 ml polypropylene tube.

c. Divide gel slices exceeding 400 mg among additional tubes. Add 60 μl of Gel solubilization buffer (L1) for every 10 mg of gel.

3. Gel solubilization

Incubate at 50 ºC for > 15 min. Mix every 3 min to ensure gel dissolution. After gel slice appear dissolved, incubate for 5 min longer.

4. Cartridge loading

Place a spin cartridge into a 2 ml wash tube. Pipette the mixture from step 3 into the spin cartridge. Centrifuge the mixture in a microcentrifuge at > 12,000 rpm for 1 min. Discard the flow through.

Note: Load no more than 400 mg agarose per cartridge.

5. Optional cartridge wash

Place the spin cartridge back into the 2 ml wash tube. Add 500 μl Gel Solublization Buffer (L1) to the spin cartridge. Incubate at room temperature for 1 min, then centrifuge at > 12,000 rpm for 1 min. Discard the flow through.

6. Cartridge wash

Place the spin cartridge back into the 2 ml wash tube. Add 700 μl of Wash Buffer (L2) (containing ethanol) to the spin cartridge and incubate for 5 min at room temperature. Centrifuge at > 12,000 rpm for 1 min. Discard the flow through. Centrifuge again for 1 minute to remove residual wash buffer.

7. DNA elution

Place the spin cartridge into a 1.5 ml recovery tube (supplied). Add 50 μl of warm TE Buffer (TE) directly to the center of the spin cartridge. Incubate for 1 min at room temperature, then centrifuge at > 12,000 rpm for 2 min.

Phenol-chloroform precipitation of PCR product

Add dH₂O (50 μl) in reaction mixture (50 μl) to make up volume to 100 μl.

1. Add equal volume phenol : chloroform (100 μl) and mix it.
2. Centrifuge for 6 minutes
3. Take supernatant in fresh tube
4. Add 3M sodium acetate 1/10^th (8 μl pH 5.2) and 2.5 volume absolute ethanol (300 μl).
5. Incubate at –20 ⁰C for 30 minutes.
6. Centrifuge for 10 minutes.
7. Dispose ethanol.
8. Wash the pallet with 70% ethanol (100 μl), spin for two minutes.
9. Dry the pallet and dissolve in 15 μl dH₂O.

Preparation of electro-competent cells of E. coli

1. Pick a single colony from a freshly grown plate of E. coli, transfer into 100 ml LB medium in 1 liter flask and overnight incubate at 37 ⁰C with vigorous shaking.

2. Take 2.5 ml of overnight grown culture and transfer to 250 ml LB in 1000 ml flask and shake vigorously at 37 ⁰C until it reach O. D⁶⁰⁰ of 0.5-1.0 (10¹⁰ cells/ml).

3. Then transfer the cells aseptically to sterile disposable 50 ml propylene tube.

4. Keep the culture on ice for 10 minutes.

5. The cells were pelleted by centrifugation at 5000 rpm at 4 ⁰C for 15 minutes and resuspended in one of volume sterile cold distilled water.

6. Pallet the cells by centrifugation at 5000 rpm at 4 ⁰C for 15 minutes and resuspend in 0.5 volume of sterile cold distilled water.

7. Again pellete the cells by centrifugation at 5000 rpm at 4 ⁰C for 15 minutes and resuspend in 0.02 volume of sterile cold distilled water.

8. Once again centrifuge at 5000 rpm for 15 minutes and suspended finally in 0.002-0.003 volume sterile 10% cold glycerol.

9. Store the cells in aliquots of 50 μl or 100 μl at -70 ⁰C.

Transformation in E. coli by electroporation

1. Electroporation cuvetts 2 mm gap were placed on ice.

2. Thaw the frozen electrocompetent cells of E.coli on ice.

3. Add 2μl of ligation mixtures into Eppendorf tube containing the competent cells and mixe gently with the pipette tip.

4. The conditions for electroporation are:

Choose mode T 2.5 KV

Set resistance R R5 (129 ohm)

Chamber gap 2 mm

Set charging voltage 2.45 KV

5. Transfer the electrocompetent cells containing the ligation mixture to electroporation cuvette.

6. Give the electiric pulse and add 1 ml of liquid LB medium immediately, mix gently and transfer to a 1.5 ml Eppendorf tube and incubate at 37 ⁰C for 45 minutes with vigorous shaking.

7. Spread 100 μl of transformed culture on solid LB medium having antibiotics.

8. As transformed mixture absorbed completely, seal the plates with sealing film and keep at 37 ⁰C in incubator for over night.

9. Pick single clonies with sterile toothpicks and culture in 3 ml liquid L.B medium containing antibiotic(s).

10. keep culture tubes at 37 ⁰C in water bath for over night with vigorous shaking.

11. Isolate plamid (protocol described above) and verify on 1% agarose gel.

12. Restrict the isolated plasmid DNA suitable restriction enzymes for screening.

13. Mark confirmed colonies, preserve in 50% glycerol and stored at -70 °C for further use.