TOPO TA cloning - follow-up to earlier question (Jun/28/2007 )

hi...asinine question, but i'll go ahead anyway....

in the depths of my freezer i found a TOPO TA pcrII dual promoter kit from 2005. did a fresh pcr, incubated with TOPO vector for about 30 minutes, and transformed 5uL of the reaction into TOP 10 competent cells according to Invitrogen protocol. plated the entire reaction on freshly-poured Amp plates on which i had spread fresh-made X-gal in DMF. the X-gal formed a slight white precipitate on spreading but i've read that that shouldn't matter too much.

this morning i found about 15 tiny white colonies on each of my plates (i had 4 different reactions). as we speak i'm growing them up, and will perform a restriction digest on them-- this is a subcloning experiment so my PCR primers had restriction sites on them and i intend to cut with those enzymes and see if my insert's in there.... at this point i'll try anything

i guess i'm just wondering what the odds are of my insert being in those colonies. this kit is old--the topoisomerase may have fallen off the linearized vector and the vector may have ligated on itself (with my luck that's prolly what happened). the Amp plates i poured had 2.5 times the normal amp concentration (100ug/ml). any other ideas? at least this gave me some hope-- enough to sleep tonight. <bites fingernails obsessively>

thanks!

well best of luck.

If the plasmid religated to itself it should look blue.

The import bit of info is how many blue colonies did you see in comparison to the number of white colonies.

You need only 1 colony.