TB（transformationbuffer）法超级感受态制备

TB (transformation buffer) 法超感制备 Procedure (250ml)

1. Streaked DH5α on an LB-agar plate, and incubated at 37 °C overnight

2.Pcked 10 large colonies Grow in 250ml \"SOB\"/1000ml flask at 18～23oC until

OD600 = 0.6.

3.Transferred the culture to a centrifuge tube and kept it On ice for 10 minutes.

4.Spin at 2,500g for 10 min. at 4℃.

5.Resuspend cells gently in 80 ml of ice cold \"TB\". 6.On ice for 10 minutes.

7. Spin at 2,500g for 10 min. at 4℃.

8. Resuspend cells gently in 20 ml of ice cold \"TB\". 9.Add DMSO to a final concentration of 7% (1.4ml).

10. Divided the bacteria on ice 15 minutes, then froze them in liquid nitrogen, and

stored them at -80°C liquid precursor CaCl2 3 M KCl 3 M Pipes 1M pH6.7 MgCl2 2 M MnCl2 1 M

SOB Medium （1L） bacto tryptone 20 g yeast extract 5 g NaCl 0.5 g

KCl 0.83 ml （3M stock） After Autoclave

MgCl210 ml （2M stock） TB 200 ml 500 ml Pipes 10 mM 2 ml 5 CaCl215 mM 1 ml 2.5 KCl 250 mM 16.7 ml 41.7 MnCl2 55 mM 11 ml 27.5