Magenta Faun
posted over 4 years ago

about PC105 dLwCas13a-GFP plasmid from addgene

I ordered the PC105 dLwCas13a-NF (supposed to contain GFP and SV40 NLS) plasmid from addgene. There are two problerms when I work with it:

(1) When I tried to expand them with stand miniprepe protocol, the yield is pretty low and very easy to get a 2-3kb band, which seems there is recombination.

(2) I transfected the plasmid again to Stl3 strain, did the prep. and validated with enzyme digestion. This time I get enough plasmid. However, when I transfected the plasmids to 293T cells, there are a lot of cytoplasm GFP signal instead of nuclear localization.

Does anyone see this happen before? Any suggetions to deal with it?

Mint Lichover 4 years ago

If there is recombination Stbl3 is a good choice, which you already did. However, pUC15 is not based on a lentiviral backbone so it should not recombine a lot.

As for the GFP signal, did you just observe cytoplasm or cytoplasm plus nuclear localization? Overexpression of the protein can lead to "leaky" cytoplasm signal.

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