Help with ligation intoIMPACT-CN pTyb11! - (Nov/19/2001 )

Please help -- I have been trying over and over again to ligate a 300 bp sequence I have PCR'ed up with engineered Sap1 and Xho1 sites. The PCR product was cloned into the TA pCR2.1 vector from Invitrogen to allow for easy cutting of the Sap1 site. I am able to visualize my cut insert and vector on a low-temperature gel, which is folled by phenol-chloroform purification. Standard ligation using T4 DNA ligase from Gibco is done overnight at 16C. However, upon transformation, no colonies with insert are obtained! We have been trying this for quite some time, and deadlines are approaching! Thanks in advance, Chris Sturgeon

Chris

I have used this system myself.... I usually do a gel purification (Qiagen kit) instead of a phenol extraction. I have had no troubles with the ligation or transformations. Actually, there was one protein that I was cloning that was hard to ligate. After the transformation I had to plate as much of the volume as I could (~400ul) to get even a few colonies. Good luck!