Hi there
I'm planning on annealing two PCR products to perform some mutagenesis in a plasmid. I was recommended to try and keep it to 600pb each, for example, but for one of the plasmids there are no enzymes avaliable at less than 1300pb. Annealing two PCR products of 1300 each with 25-30 pb shared sequence is too much? Will it work? Will it be too much to then introduce it into the plasmid?
Please help me
Bea
Hello Beatriz,
I don't have idea of the maximum bp that can be ligated but I recently worked on a projected that I used overlap PCR to amplify 2 PCR products of 500 bp each with 15-20 shared bases and it worked fine. Bear in mind that you will need to gel purify your overlap pcr product that corresponds to your expected size.
Best wishes in your experiment
Hello Beatriz,
I don't have idea of the maximum bp that can be ligated but I recently worked on a projected that I used overlap PCR to amplify 2 PCR products of 500 bp each with 15-20 shared bases and it worked fine. Bear in mind that you will need to gel purify your overlap pcr product that corresponds to your expected size.
Best wishes in your experiment
Annealing PCR products of more than 1kb size with overlapping/sharing of 15-20 shouldn't be a problem as long as the molar ratio used is correct. I've tried cloning pcr products of more than 1kb into plasmid using hot fusion method without any problem.
Article Hot Fusion: An Efficient Method to Clone Multiple DNA Fragme...
Hope this helps, good luck!
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