Difference between revisions of "Team:REC-CHENNAI/Protocol"

Revision as of 16:32, 28 October 2017

SOB

Ingredients

0.5% (w/v) yeast extract
2% (w/v) tryptone
10 mM NaCl
2.5 mM KCl
20 mM MgSO₄

Per litre

5 g yeast extract
20 g tryptone
0.584 g NaCl
0.186 g KCl
2.4 g MgSO₄

Note: Some formulations of SOB use 10 mM MgCl₂ and 10 mM MgSO4 instead of 20 mM MgSO₄.
SOB medium is also available dry premixed from Difco, 0443-17.
Adjust to pH 7.5 prior to use. This requires approximately 25 ml of 1M NaOH per liter.

BUFFERS

CCMB80 BUFFER

10mM KOAc pH 7.0 (10ml of a 1M stock/L)
80mM CaCl₂.2H₂0 (11.8 g/L)
20mM MnCl₂.4H₂0 (4.0 g/L)
10mM MgCl₂.6H₂0 (2.0 g/L)
10% glycerol (100 ml/L)
Adjust pH down to 6.4 using 0.1N NaOH if necessary.
Sterile filter and store at 4^oC

Note: Slight dark precipitate may appear. However, it will not affect its function.

TAE BUFFER (pH 8.3)

Tris base – 48.4g/l.
Glacial acetic acid 11.4ml/l.
0.5M EDTA – 20ml. (3.72g for 20ml)

Note: The volume is made up to 1 litre using distilled water.

TE BUFFER (pH 8)

Trise base – 1.214g/10ml
0.5M EDTA – 2ml (0.372g for 2ml).
HCl – To adjust the pH to 8.

Note: The volume is made up to 1 litre using distilled water.

COMPETENT CELLS PREPARATION

Inoculate a loop full of culture in 5ml of LB broth and incubate it in a shaking incubator at 200rpm for 24hrs.
Inoculate 1ml of the overnight culture in 250ml of LB broth.
Incubate the broth containing the culture in a shaking incubator at 200rpm at 37^oC until the OD610 reaches 0.3-0.6.
Freeze the culture at 4^oC for 30minutes to arrest the metabolism.
Then aliquot the culture equally into 15ml falcon tubes.
Centrifuge the tubes at 6000rpm for 10minutes. Discard the supernatant and collect the pellet.
Add 4ml of chilled CCMB80 to the pellet and resuspend them gently using a pipette.
Centrifuge the tubes at 6000rpm for 10minutes. Discard the supernatant and collect the pellet.
Resuspensd once again using 2ml of chilled CCMB80.
Centrifuge the tubes at 6000rpm for 10minutes. Discard the supernatant and collect the pellet.
Resuspend the pellets using 1ml of chilled CCMB80.
Centrifuge the tubes at 6000rpm for 10minutes. Discard the supernatant and collect the pellet.
Resuspend the pellet in 100µl of chilled CCMB80 and combine the cell masses in all the tubes.
Thus competent cells are prepared using CCMB80 buffer.

Note: Resuspension and centrifugation processes must be performed at 4^oC to produce efficient competent cells.

TRANSFORMATION

Add 50µl of competent cell culture to pre-chilled 1.5ml vial.
Add 1µl of given plasmid DNA to it and incubate in ice for 30minutes.
Heat-shock the cells by placing the vials in a water bath at 42^oC for 90seconds.
Place the vials in ice for 10minutes.
Add 600µl of LB broth and incubate the culture in a shaking incubator at 200rpm and 37°C for 2hours.
Plate the culture on chloramphenicol plates and incubate the plates at 37^oC

Note: Colonies will be formed only after 24-36hrs of final incubation.

Click here to download the PDF

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