pEGFP C-1/N-1 Cloning - (Jan/11/2013 )

Hi all,

I am new to the forums, but I have been a longtime lurker when the need arose. I am having some difficulty cloning a 1kb insert into the pEGFP vector (Kan resistant).

I know my ligated product is present via PCR and by simply running it on a gel. My problem is when I transform my plasmid, I get zero colonies. I have tried multiple methods of transformation with a variety of cells. Commercially competent one shot, sure2, XL-10 gold, able k, and also lab produced DH5alpha and DH10Beta. I will usually use 50-100ul of cells with 1-5uL of ligated product (depending on concentation, with max being 50ng). I will allow cells to incubate for 30min, heat shock for 45 sec at 42C (or commercial competent suggested duration/temp) and allow cells to recover for 2-5 min on ice. I then rotate cells at 37C for 1-3 hrs and plate.

I have tested by original plasmid (decent colony number) + my linear plasmid (no colony number) and everything seems fine.

My only idea would be to transform on a Kan- plate to see if my Kan resistance marker was somehow damaged during cloning, but doesn't seem very viable.

Can someone offer a suggestion???

Thanks.