Hi, I am trying to perform a ligation of PCR products in plasmid digested with BbsI and purified from gel with Qiagen kit. The day after transformation i didn't have any colonies and my doubt is regarding the ligation process...
The yeld of purified plasmid from gel was 30ngr/ul but with high levels of EtoH (nanodrop measurment). So my question is: Is it possible that EtoH can inhibit the ligation reaction? I used 2ul of plasmid in 20ul of final volume of ligation performed with T3 enzyme.
In case it is possible, do you know how I can purify the digested plasmid from etoh? speedvac could be an option but I am worried to lost the low amount of DNA
It's quite easy. Just do two experiments side by side: one with EtOH, and the other with the same volume of distilled water instead of EtOH. And viola, you have the answer right away.
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