I need to transform E. coli TAP56 with a plasmid of 5.6 kbp. The CaCl2 transformation protocol was working fine earlier. But for last few weeks this method is not working at all. We have sequenced our plasmid and found to be ok. I have tried with several variations in the protocols as well, like, change of plasmid concentration, change of heat shock temperature, change of CaCl2 concentration, adding 2-mercaptoethanol to remove interfering proteins. It did not work! What can I do to overcome this problem?
I didn't try with any positive control plasmid! But the method works fine with a different plasmid pRE2 of 4.8 kbp.
I add CaCl2 to cells and take 50 microliter in eppendorf tubes. Keep them at 4°C for 22-24 hours. Then I am following standard CaCl2 transformation protocol. Making the cells competent for a longer period seems work fine!!!!
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