Unstable plasmid ?? - Deletions only after ligation ! (Feb/22/2005 )

Hi,

happy to find this forum as I have a big problem with a plasmid I use (actually the most important as my whole project will be based on that for the next 3 years )

The problem is that the plasmid seems to be very unstable at least in the conditions I use ! When I transform any strain of e-coli with the original plasmid all the colonies after miniprep and digestion seem to have the correct plasmid. The problem comes when I'm trying to put an insert in the plasmid ! The result I get from every colony is different but none has the correct restriction profile (everyone with different deletion) ! I've tried culturing at 30 and 37, transforming strains like DH5alpha, XL10 Gold, SURE and all give same results ! I 've tried also a second insert ... again with same problems and even worse ... returning from all the minipreps the same plasmid much smaller than the original giving a single band (the original has 5) after the digestion .
(No contaminations)

It seems like my bacteria are playing with my plasmid and my lack with me and my PhD. .....

Hiya!

Check your insert and plasmid for restriction sites....you might have some sneeky ones slithering around...mocking you...... Or your enzymes might have some star activity.....in that case they are mocking you...... Re-evaluate your digestion protocol........come on.... you're the eukaryote.....you show'em whose boss!!

Hope that helps
Labprincess

Everything is fine with restriction sites !
The problem is with the deletions (only when I put an insert !)