double digested PCR product into competent cell - (Jan/31/2007 )

Hi everyone, could you help me look at what may be the problem ?

1. I have cloned a fragment of ~700bp PCR product flanking with NheI and Hind3 from human genomic DNA, 3 extra base-pairs used at the end for the restriction enzymes. Then the products were kept at -20degree before use.

2. pGL3-basic vector was chosen. Double digestion was done for both plasmids and PCR products, 37 degree 3 hrs. Then pGL3-basic was gel purified, PCR product(without running on gel) was also purified using Wizard SV gel and PCR clean up system. Double cut pGL3-basic and PCR product, single cut pGL3-basic with either NheI or Hind3 were run on 0.8% agarose gel, no problem with enzyme digetion.

3. Ligation was done. vector:insert=1:3 used. 4 degree, ON. Meanwhile, double cut, not purified pGL3-basic and purifed pGL3-basic was ligated for liagation step control.


4. Ligated mixture was transformed into competent DH5 alpha, and some controls were done. The result was no problem for ligation and transformation step. There was also no reliagtion for purified pGL3-basic vector.


I have wondered whether it due to the unable digestion of PCR product, but HOW can I check and WHAT step can be modified?

Thanks very much for help~~~

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Err.... what is the major problem? It seems to me that your ligation step works. Meaning, I am sure you did check your ligated product via direct PCR, enzyme digestion and sequencing?

Mind explain further?

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The problem is there was no colony for vector ligated with the PCR products.

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