2 replies to this topic

[jungy1]

Hello there,

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??

[phage434]

Possibilities:
* 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)

[pDNA]

it could be "ghost"-bands that you have seen during colony PCR ...they often appear as faintly bands on your agarose gels ...mimicking positive clones.
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!