pBluescript - (Sep/17/2004 )

HI,

Have you used the plasmid pBluescript? I am trying to clone my insert into the BamHI site of pBluescript. After performing the ligation reactions, I transformed E. coli Top10 bacteria, and I got zero colonies, even on the self-ligation plate.
I was wondering if the strain of bacteria might be the problem i.e TOP10 is not suitable for pBluescript?

Thanks for reading.

To be sure your competent cell is good or suitable for pBluescript, always do a positive control using intact plasmid. You said your self-ligation plate didn't grow colonies either, so you used BamHI-cut-an-then-ligated pBluescript. In that case, the ligation reaction could be another source of failure. Just use the uncut one.

Hi,

I also used pBluescript SK- for many clonation stuff. If i get it correctly from your description, you have only cut with BamHI in SK-. Then you have to make a SAP treatment with your vector. You also have to know what is the concentration of the plasmid and your fragment after purification. Then you can make variations of Ligation reactions. Maybe take the Rapid Ligation Kit Boehringer. for the transformation take dh5alpha (e.g. invitrogen). It works very well with SK-, normal trafo, heat shock, 2ul lig in 35 ul comp cells, 20 min ice, 30 sec 42 C°, 2 min ice 1 hour 37C° shaking and spread all(300ul) on Amp plates. It must grow colonies! And make always positive and negative controls for ligation and trafo

Good Luck