Trying to sub clone a 300 bp gene in pET11a vector with Nde1 and BamH1 R.E . I'm getting a positive colony PCR and Re-PCR however on double digestion with the respective enzymes no fall off is observed. I've repeated all the experiments thrice, even changed the enzymes,buffer and incubation time but still left with the same result. Please suggest
Hi,
We also face the same problems many time. Many times PCR can provide false result as I observed the amplification of insert but not in RE.
I totally agree with above suggestion and you can use the other combinations of RE which cut at any portion of vector and compared with empty vector and look for difference of 300bp.
If you want to put new ligation then use very little amount of vector, because high concentration of vector gives many colonies with vectors only.
Your primer have template contamination. U can check it through a PCR reaction without using any template.
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