1.The single colony of E. coli was inoculated in 2ml SOB medium, and shaken overnight at 37 ℃.
2.Transfer 0.5-1ml of overnight culture broth to 50ml of SOB, shake violently at 18 ℃ until A600 reaches 0.6.
3.The culture was transferred to a 50ml centrifuge tube and centrifuged at 4 ℃ 4000rpm / min for 10min. At the same time, TB solution is prepared on the ice bath.
4.Discard the supernatant, put the centrifuge tube upside down on the filter paper, and let the culture solution be dried.
5.Take 1ml of TB solution just prepared to disperse the bacterial precipitation, and then add 15ml of TB(1 / 3 volume of initial medium),ice bath 10-15min, 4 ℃ 4000rpm / min centrifugation 10min.
6.Discard the supernatant and suspend the sediment to 4ml TB 1 / 12.5 volume of starting medium，ice bath 10-15min.
7.Add 280 μl DMSO, drop it in slowly and shake it gently to make it mix well, and take an ice bath for 10min.
8.Divide the bacterial solution into EP tubes and store it in - 80 ℃ or liquid nitrogen.
9.Add 1 μl sterile ddH2O to the two tubes of receptive cells(negative control) and transformation of 1 μl pure plasmid (positive control),to detect the quality of the sensory state.The negative control plate should grow aseptically,the number of colonies on the positive control plate shows the sensitivity efficiency.
Preparation of SOB:
| peptone yeast extract NaCl KCl 100×Mg++ solution |
20g 5g 0.58g 0.186g 10ml |
Dissolve and add water to a constant volume of 1l, and sterilize with 121 ℃× 20min high-pressure steam.
100×Mg++ solution:
MgCl2﹒6H2O
MgSO4﹒7H2O
Preparation of TB solution:
| 1M KCl 0.55M MnCl2 0.5M CaCl2 0.1M K-Pipes(pH 6.7) ddH2O Total |
5ml 2ml 0.6ml 2ml 10.4ml 20ml |
All the above solutions need to be sterilized by high pressure steam.
Preparation of 0.1mk-pipes (pH = 6.7)
Weigh 3.02g pipes powder and dissolve it in 80ml DD H2O. At this time, the powder can not be completely dissolved. Adjust the pH value with 10nkoh or KOH solid. The powder can be completely dissolved only when the pH is close to 6.7. At this time, carefully add KOH in small amount until the required pH value is reached.
Related reading：
preparation of yeast media
Expression Of Protein In Escherichia Coli