Hello All,
I'm trying to clone a 1.5 kb fungal gene to a PJET cloning vector (Thermo scientific) that has 98% selection capability, and I'm also verifying the clone using the PCR, but every time after sequencing, I get the result as a vector sequence rather than the specific clone fragment that I'm targeting. Please suggest me if any one aware about this problem?
Did you verify the clone by the external pJet Primers or your gene specific primers ? I use this kit ( 10 ul version (1/2 protocol) only) regularly and still there are some background clones arising sometimes.But usually it works well.Important control versus PCR control and your fragment, which gives a good indication of the working experiment.Then I perform < 30 min classical restriction analysis.
Hello Sumit, more information would help us troubleshoot. Can you kindly attach your protocol? Alternatively , you can try RecA-independent recombination or RAIR cloning, see publication below. You just need four primers—two to amplify the vector and two to amplify the insert— where the 5’ and 3’ ends (of the resulting PCR amplicons) are homologous to one another. Following PCR, you just mix 1-2 microliters of each reaction to heat shock cells, perform the heat shock protocol, plate the cells, and perform colony PCR the next day to select the positive clones. Most common laboratory strains of E. coli are capable of RAIR. Good luck!
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