Protocols

Preperation of competent DH5alpha cells

Autoclave 0.1 M CaCl2 in 20% glycerol.
Primary culture in 5 ml of LB broth for 16 hours.
Add 1 ml of primary culture into 50 ml of media for secondary culture.
Check OD at 600 nm.
Incubate secondary cultures at 37 C in a shaker.
Check OD600 after 1 hour, 1.5 hours, 2 hours.
Keep the cells on ice (for 10 minutes) for harvesting as soon as OD600 reaches 0.4 (0.4 -0.6 is the range).
Pellet down the cells at 6000 rpm for 10 min (4 C ).
(Each 50 ml culture can be added to 50 ml falcon tubes)
Add 10 ml of ice cold 0.1M CaCl2.
Resuspend the pellet and keep on ice for 10 minutes.
Centrifuge at 6000 rpm for 10 min (4 C).
Resuspend the pellet in 5 ml ice cold 0.1 M CaCl2 Solution.
Again, keep on ice for 10 min. and centrifuge at 6000 rpm for 10 min.
. Resuspend the pellet in 2 ml ice cold 0.1 M CaCl2 Solution. Alliquot 100 ul into a number of tubes and store them at -80 C.

Transformation

Miniprep using alkaline lysis buffers

Agarose Gel Electrohoresis

Restriction digestion

Gel Elusion

PCR

PCR_purification

Ligation

Team:IIT Madras/Notebook/Protocols