plasmid size change after transformation - (May/09/2006 )

Hello,

I received a plasmid from a colleague & transformed some E.coli (invotrogen TOP10) to get my own stock.

question 1: when I run the two plasmids on a gel side by side, they look similar (three bands) but the original plasmid runs overall larger than my new stock. Anyone got any ideas about this ??


question 2: I'm double digesting (BglII + XhoI) the plasmid. The aim is to ligate in some oligos that have complimentary overhangs. I'm CIAP treating the vector & gel purifying before ligation. I have PCR primers that in empty plasmid give 330bp, and with the insertshould give 390bp.

when I ligate & transform bacteria, then perform colony PCR, I get all negative (330bp) results. Does this indicate empty plasmid transformation ? How did it get there after CIAP treatment & gel pur ?

thanks !!!

Hi

Not sure why in regards to the plasmid size but I have seen it as well. Are you running uncut plasmid or are you linearizing it? You may want to digest both your transformed plasmid and the original stock with some restriction enzymes and check that you are getting the same banding patterns.

As for the empty inserts - you're probably getting empty plasmids because your CIAP treatment is not 100% efficient. I've seen other people posting about this on the forum. A lot of people seem to advocate SAP instead of CIAP because it's easier to heat inactivate but also some people believe it gives better de-phosphorylation results.

Hope this helps!