Problems ligating

Rhoda Deguzman deguzman at utsc.utoronto.ca
Sat Jun 16 17:00:16 EST 2001


Hi,

I'm having trouble ligating sticky ended (XhoI) genomic fragments to
bluescript KS II (+/-).  I'm using XhoI fragments ranging from 4 to 8Kb
in order to isolate my 5kb band of interest.  My insert and vector are
cut,and clean.  I treated the vector with shrimp alkaline phosphatase
(SAP) and all enzymes have been inactivated according to manufacturer's
specification.  My controls show that SAP, and T4 ligase are working.  
However, when the reactions with insert and vector are examined on the
gel there is high molecular weight bands not previously present but the
vector band is still very prominent.  It's intensity is close to the
amount initially added, so it makes me wonder if the high molecular
weight bands are just recircularizing inserts. Please help I don't know
where I'm going wrong. I've played with different ratios of insert to
vector, using 1:1 upto 1:4 and still there are very few white colonies. 
My transform my cell by electroporation and I don't think the problem is
there.  Any suggestions would be greatly appreaciated.

Thanks
Rhoda

deguzman at utsc.utoronto.ca

---




More information about the Methods mailing list