Hi. For gene deletion, I need huge quantities of highly concentrated linearized plasmid for electroporation, but, after restriction digest, I have hard time to recover satisfying quantities by ethanol or isopropanol precipitation (plasmid starting material used in restriction digest as well as linearized DNA recovered have been dosed using Qubit). Does anybody have some suggestions ?
Dear Willy. I think there is no reason why the DNA yield should be low. So, the problem should be more likely in ethanol/IPA method. Please check that (eg, use glycogen, spin more time, don't disturb the blue glycogen precipitate). Or, the yield is good but due to some substance precipitated, the DNA concentration looks to be low but its actually its enough. (May be run in gel and see if DNA is actually low)
Improving DNA precipitation yield after restriction digest involves optimizing several steps in the process to maximize DNA recovery. Here are some strategies you can consider:
By implementing these optimization strategies, you can improve DNA precipitation yield after restriction digest and obtain higher-quality DNA samples for downstream applications such as cloning, PCR, or sequencing.
add salt (NaCl or ammonium acetate (1/10volume of a 5M solution ph5) before adding 2 volumes of cold ethanol