1． Put the competent cells into ice box as soon as their solution melts.
2． Add ligation system 10uL or plasmid 0.5uL into solution.
3． Keep in ice for 30min.
4． Heat shock in 42℃, JM109 and DH5alpha for 30s, BL21 for 120s, all other for 90s.
5． Put the competent cells into an ice box immediately for 2min.
6． Add 800uL LB media in a super clean bench.
7． Recover 160rpm for 30-45min.
8． If it is positive plasmid, smear 100uL solution directly onto plate. If it is ligation system, centrifuge for 5000rpm for 4min, discard the supernatant and smear the remainming onto the plate with antibiotic medium.
Preparation of Competent Cells
Bottles, tubes and tips
Yeast Extract 5g
Add water to form a homogeneous solution
1L SOB + 10mL 2Mg2+ (1M MgCl2 + 1M MgSO4)
First time activation
Pick 1-10 single conlony from the plate
Add SOB，37℃ cultivated until saturation
PIPES 3.0g 10mM
CaCl2.2H2O 2.2g 15mM
KCl 18.6g 250mM
5N KOH -> pH 6.7
MnCl2.4H2O 10.9g 55mM
+ddH2O filtering the bacteria
Second time activation
18℃ cultivated overnight
OD 0.4~0.8 (0.6 optimal)
centrifuge, tips, TB, tubes, pre-frozen tubes
ice for 10min
1/3 v/v TB resuspend，ice for 10min
centrifuge for 1000rpm 10min
discard the supernatant, add 1/12.5 v/v TB resuspend
Add 7%v DMSO, ice for 10min
100uL/tube, quickly frozed using liquid nitrogen under -70℃