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Help needed for Ligation/Transformation - (Jun/24/2014 )

Hi, i am doing a mutation by deleting a certain part of the gene (deletion part is ~1kb).

therefore i need to ligate the upper and down stream into my vector pUC19 (~2.6kb).


The first (upper stream piece) ligation and transformation turned out to be good but everytime i try to ligate the second piece (the down stream) into the vector, it always failed.


PS. 1. Both upper and down stream are around 1kb and they were purified after pcr; plus i also tried to use the insert after gel extraction but didn't work either.

       2. Upper stream restrction enzymes are PstI and XbaI. Both vector and insert were digested and the vector were  treated with CIP before ligation.

      3.Downtream side are XbaI and BamHI, double-digested vector(whch is the product after upper stream ligation) and insert. Vector and insert ratio is ~3:1 but this step always failed. 


Does anyone know how to solve this??? Thank you!


I would blame cip, which never works for me. Switch to shrimp alkaline phosphatase or antarctic phosphatase, and heat kill it. CIP can't be heat killed, and you need to remove it before ligation.

Another possible problem could be toxicity of your final product. Could your construct be making a toxic gene?