jerryshelly1

Not really sure if this belongs here.  It just seems like this CutSmart buffer appeared over night.  Is this a replacement for the EcoRI buffer?  Has anyone used it?  It is reporting 100% activity in a double digest that NEB previously showed incompatible (my previous reactions).  Can anyone confirm this?

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.