REMEMBER TO INCLUDE A TRANSFORMATION CONTROL. i.e. 0.1ng supercoiled vector Should give~100 colonies. Also all Ligation/SAP controls.
1) Pour LB agar/AMP plates -Dry 20mins in cell culture hood & warm. If you store plates return them to the plastic sleeve and indicate name date and antibiotic used.
2) Thaw 50 l competent cells on ice - Use only Rec A- starins JM109 or DH5a.
3) Immediately add 1.7l of 1/10 dil B-mercaptoethanol. Ice 10mins
4) Add 2l or 5l not more! of ligation. Mix gently and ice 30mins.
5) Heat shock 2mins at 42oC
6) Ice 2mins at 42oC
7) Add 500l warm SOC - no antibiotics! and incubate 37oC 1hr.
8) Plate 200/plate. Incubate o/n 37oC
3) In an eppendorf tube add :
1l (1ng) of plasmid DNA (or 3-5l of the ligation)
+ 99l (or 95 l) of MgCl2 30mM-CaCl2 50mM
4) Add 20 l of competent cells (or 100 l). Mix gently and let on ice 30 mins
Then go to step 6 above