extracting custom library plasmid

I made a custom sgRNA library following Joung et al. I did the transformation and collected the colonies from the plates. I was wondering if I can pool all the ecoli pellet, then pick up 3gr of the pellet, do plasmid extraction (3gr fro 1 column of maxi prep kit ) and send it for NGS. I would like to make sure the sequences are OK before going any further.


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Sienna Selkieabout 2 years ago

Yes, you should pool the pellets or do maxipreps from several pellets and pool the isolated plasmid. There's no need to maxiprep the whole pellet as it will be too much plasmid, but it's important that a part of pellet from each plate is maxiprepped. Only I think 3 g per column is too much, don't know which kit you are using but for Qiagen around 0.5 g is maximum.

Mint Baloralmost 2 years ago

Thanks a lot for your great advice.

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