I am trying to clone two genes in pET 28a vector using BamHI and XhoI sites and BamHI and EcoRI sites respectively. I am purifying the PCR product before digestion using Qiagen kit, but in the process I am loosing a lot of DNA.
Is it absolutely necessary to purify the PCR product before digestion? If yes, is there any other way (besides using the Kit) which can give higher yields?
Please help.
Thanks
yeah it is necessary to purify the PCR product before going for digestion with enzymes, in my lab we are using Mini spin colum tubes for purifying the PCR product
My approach is as Sa's: clone the PCR product into a TA vector (like pGEM T-easy, using their protocol).
Sorry, I didnt read your question properly! If you have a good yield of PCR product (according to gel analysis) you can try precipitation with 2 vols of ethanol (plus 1mg of glycogen if your PCR yield is low). That will remove salts, triphosphates and most of the primers. Then dissolve in TE, check by nanodrop and away you go!
Yes, purification is an important step. Contaminants could be a big problem on your result.
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