Hello,
I am working on some gene manipulation experiments; many times I faced a problem, When i t-clone my GOI(Gene of Interest) if give many colonies. When i run Colony PCR from the middle portion it gives exact sized positive result, but when I try to amplify full fragment, it gives nothing. When I mini-prep it and double digest it, I dont get any band of vector size nor fragment size. I did this about 10 times, What may be the problem? It is happened when I am using LA taq Takara.
i would try pcr from vector into each end of your GOI and the larger vector to vector across the goi just in case there are pcr mutations in the ends of your goi ( particularly if you had to use low annealing temperatures to generate your cloned sequence) that stop the specific primers from annealing and working. If these amplifications work then you will need to amplify other colonies or sequence the amplified ends of your goi to see why the primers are not annealing
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