Transformation - (Jun/28/2007 )

I have problems in cloning PCR product( TOPO-cloning):
I incubate the ligation mixture at 20C for 30 min, and then add 2ul to the TOP10 cells and transform. I get very few clonies about 50-60 on each plate. The transformation efficiency is low and most of the time there is no insert. The size of the PCR fragments are 500bp and 3Kb. The amount of PCR product per ligation for each fragment is 40ng.Any help would be appreciated.
Esi

Do you think 50-60 colonies are few? Isn't that more than enough? (It is for me anyway).

Dont you need only 1 colony that has your clone, that will be enough right?

50-60!!!!you have more than enough. just take a few per plate, make a mini prep. digest some of the plasmid. better if you have an enzyme that cut in 2 sites. It will be easier to know if you have the insert or not.

50-60, my old PI would have made me pick them all and look for insert.

One right clone is all that you need.

Don't go by what the manual says as to the number of colonies you would get. It's OK to have 50-60 colonies. In any case, you need to inoculate only 1 colony. Look for a large colony on your plate. Use that for mini-prep. That will likely have your insert. The smaller ones usually don't have the insert.