Team:REC-CHENNAI/Protocol
PROTOCOL
stars 10mM KOAc pH 7.0 (10ml of a 1M stock/L)
stars 80mM CaCl2.2H20 (11.8 g/L)
stars 20mM MnCl2.4H20 (4.0 g/L)
stars 10mM MgCl2.6H20 (2.0 g/L)
stars 10% glycerol (100 ml/L)
stars Adjust pH down to 6.4 using 0.1N NaOH if necessary.
stars Sterile filter and store at 4oC
stars Tris base – 48.4g/l.
stars Glacial acetic acid 11.4ml/l.
stars 0.5M EDTA – 20ml. (3.72g for 20ml)
stars Trise base – 1.214g/10ml
stars 0.5M EDTA – 2ml (0.372g for 2ml).
stars HCl – To adjust the pH to 8.
stars Inoculate a loop full of culture in 5ml of LB broth and incubate it in a shaking incubator at 200rpm for 24hrs.
stars Inoculate 1ml of the overnight culture in 250ml of LB broth.
stars Incubate the broth containing the culture in a shaking incubator at 200rpm at 37oC until the OD610 reaches 0.3-0.6.
stars Freeze the culture at 4oC for 30minutes to arrest the metabolism.
stars Then aliquot the culture equally into 15ml falcon tubes.
stars Centrifuge the tubes at 6000rpm for 10minutes. Discard the supernatant and collect the pellet.
stars Add 4ml of chilled CCMB80 to the pellet and resuspend them gently using a pipette.
stars Centrifuge the tubes at 6000rpm for 10minutes. Discard the supernatant and collect the pellet.
stars Resuspensd once again using 2ml of chilled CCMB80.
stars Centrifuge the tubes at 6000rpm for 10minutes. Discard the supernatant and collect the pellet.
stars Resuspend the pellets using 1ml of chilled CCMB80.
stars Centrifuge the tubes at 6000rpm for 10minutes. Discard the supernatant and collect the pellet.
stars Resuspend the pellet in 100µl of chilled CCMB80 and combine the cell masses in all the tubes.
stars Thus competent cells are prepared using CCMB80 buffer.
stars Add 50µl of competent cell culture to pre-chilled 1.5ml vial.
stars Add 1µl of given plasmid DNA to it and incubate in ice for 30minutes.
stars Heat-shock the cells by placing the vials in a water bath at 42oC for 90seconds.
stars Place the vials in ice for 10minutes.
stars Add 600µl of LB broth and incubate the culture in a shaking incubator at 200rpm and 37°C for 2hours.
stars Plate the culture on chloramphenicol plates and incubate the plates at 37oC
