vector problems - (Oct/23/2006 )
I have been having some trouble transforming a new insert of mine into DH5a cells. I have put the insert (about 300 bp) into a pET vector using NdeI and BamHI sites and ligated according to our labs protocal. After transformation I get colonies and after PCR of those I see that my insert is there. I then do a miniprep of those colonies and find that when I run a gel of the minipreps, to do a quick quantification of the concentration, that none of my vector is present. I have done this a couple of times following the protocal exactly how it is stated. The kit is the Qiagen Miniprep kit. So if anyone has any suggestions of why this vector is not sticking to the column it would be greatly appreciated. Thanks.
could you tell us how much culture you start with, and what volume (and of which buffer) you elute the plasmid from the column?
When you PCR, did you include negative controls? (Just to confirm that your PCR is reliable to detect your construct)
What worries me when I can't get plasmid from my colony the most is satellite cells. You could try plating or inoculating them in a higher concentration of antibiotic.
But to troubleshoot if it's the kit's problem, you could try aliquoting a small volume (0.1 ml) of cell suspension and do a quick plasmid isolation by resuspending pelleted cells in "solution I" followed by lysis in solution II (Same as Sambrooks concoction [or use the P1 and P2 as substitute]). Then resolve in agarose gel to see if your plasmid is present. Note that you'll see genomic DNA and RNA bands as well. But if you want a cleaner prep, add solution III and follow the procedure by the book.
Hope it's not satellites.