I would like to amplify two toxin genes of C. difficile followed by expressing toxin A and B using bacterial expression system. In which we started with the amplification of gene using PCR, we performed many times using different enzymes, GC buffers and time intervals, but we are not successful. Since, our gene of interests are larger in size 8kb and 7kb for toxin A and toxin B respectively, it is difficult for us to get expected result in PCR.
Q1: Our lab mates used bacterial cells directly for PCR - is this correct?
Q2: If Q1 is wrong, is it essential to purify the DNA from the cells first and perform PCR?
Chromosomal DNA is better. You need a good long range taq or will have to do it in pieces and ligate them together. Think of the host you wish to express in - gram negative backgrounds will give rise to the problem of getting rid of LPS.
Will be interested to know your progress.
http://www.biomedcentral.com/1471-2180/8/192
http://www.sciencedirect.com/science/article/pii/S0006291X03012348
Use phusion polymerase for doing PCR; and use genomic DNA as template.
https://www.neb.com/products/m0530-phusion-high-fidelity-dna-polymerase
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