problems with miniprep result - (Sep/29/2006 )
I have some problems with my miniprep results. Currently I'm trying to carry on a sequencing of a site directed mutagenesis product. To do that, I transform E coli strain DH5alpha and isolate plasmid by miniprep. I've had some problems when using Wizard SV plus miniprep kit. I normally use 10ml of the overnight culture (no OD measurement before cell lysis) to extract a high copy plasmid (I know I could use lower volumes, but I'd like to have greater amounts of plasmid). Sometimes a clot that does not precipitate forms in the microtubes. This is my first question: how do I precipitate the lysate in the clot?
Second and most important, sometimes I run an 0,8% agarose checkgel for a miniprep of clones resulting from this site directed mutagenesis PCR product and I get a band standard different from that expected. Bands are all of different sizes in all lanes (one lane for each clone prep product) thus not corresponding to one another even though plasmid extracted is supposed to be the same. What are the possible causes for this?
Just a question to help clarify matters, with respect to the aberent size bands, is the DNA linearise?