plasmid DNA disappear in the minipre
Posted 01 December 2009 - 06:03 AM
I am stucked on a big problem in minipre. The step of ligation-inserts (kpnI, xhoI) and pGL3 vector-was fine. Transformation was fine too. I shown about 200 colonyes on Amp restricted LB plate. Then I did direct PCR for confirming the inserts. I had seen inserts band too. Next I did minipre but the plasmid DNAs was disappeared..
What do yo think of this??
Posted 01 December 2009 - 06:41 AM
* you grew the cells without antibiotic and lost the plasmid
* your miniprep failed (for whatever reason)
* your pcr verification was wrong (primers bound to insert DNA left over on the plate) (but this would still give you a plasmid, just not an insert-containing one).
* your original plates had no or inadequate antibiotic and allowed colonies to grow that contained no plasmid (satellite colony formation could also be a problem)
Posted 01 December 2009 - 12:09 PM
This often happens when you pick colonies from "undiluted" ligations. Try to transfer your clones on a fresh plate containing antibiotics ...and re-do your PCR-screening.
Always include a negative control when doing ligations ...this means using vector only (no insert) + ligase to see if you get background from religation of your vector.
Doing ligations theotherway round is a wast of time because you never know what happened ...please include controls ...thats the only rational way to do it!