problem in digestion - (May/17/2011 )
friends, i have done a cloning of 300bp gene (PCR product) by TA cloning. Now I try to sequentially digest to release the insert using BamHI and HindIII enzymes. i have tried a lot but failed. I am not able to see 300bp insert getting released. when a PCR is set with plasmid as template using gene primer, i am getting exact 300 bp amplicon. now my question whether is present if yes how to release it.Thanks.
First check BamHI and HindIII digestion leads to linerisation. then do the sequental digestion, when you do the sequenctal digestion donot forget to heat inactivate the previous enzyme.