高效制备感受态细胞

时间:05-26 00:40
高效制備感受態細胞

如果用來建庫或者轉化比較珍貴的材料可以試一下这種感受態!效率非常高!

Simple and Efficient Method (SEM) to Make Competent Cells

Preparation of Frozen Competent of DH5α

1) 250 ml TB solution

10mM Pipes or 10 mM Hepes, 0.65g

15 mM CaCl2, 0.55g

250 mM KCl, 4.66g

*55 mM MnCl2, 2.47g

All the components except for MnCl2 were mixed and the pH was adjusted to 6.7 with 0.1 N HCl (If you overshoot, do not readjust the pH by adding base, instead, discard the solution and begin again). Then 2.475 g MnCl2 was added to the solution and dissolved. The solution was sterilized by filtration through a prerinsed 0.45 um filter unit. Dispense the solution into 50-ml aliquots and stored at 4°C.

2) Other Solution:

A. 100 ml of 250 mM KCl solution with ddH2O, non-autoclaved: 1.86 g of KCl in 100 ml ddH2O.

B. 10 ml of 1M MgCl2 +1 M MgSO4 with ddH2O, autoclave. (Prepare 1M MgCl2 first, then add MgSO4 into it)

C. 50 ml of 1 M glucose with ddH2O, filter, aliquot in 1.5 ml, store at -20°C

D. For 100 ml SOB medium: (Amount/volume)

Bacto tryptone: 2 g

Bacto yeast extract 0.5 g

NaCl 0.05 g

Shake until the solutes have dissolved.

Add 10 ml of a 250 mM solution of KCl

Adjust the pH to 7.0 with 5N NaOH (~0.2 ml)

Adjust the volume of the solution to 100 ml with ddH2O

Sterilize by autoclaving for 20min at 15 lb/sq. in on liquid cycle

Just before use, combine the medium with 1 ml of 2 M Mg2+ (20 mM final)

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