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Hi Peichuan,I actually had some success using Taq w/out exo...
J
Jimmy Aug 19, 2011
Hi Peichuan,
I actually had some success using Taq w/out exo ability from NEB. I saw the restriction cut product band shift ~30 bp lower than the uncut product (as well as a 30BP band faintly below). So thank you for the suggestion! Since we have a DNA synthesis core here it was Read more
I actually had some success using Taq w/out exo ability from NEB. I saw the restriction cut product band shift ~30 bp lower than the uncut product (as well as a 30BP band faintly below). So thank you for the suggestion! Since we have a DNA synthesis core here it was Read more
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Peichuan Zhang Answered Aug 19, 2011
Calico Life Sciences
Hi Jimmy,
Very glad that it worked out well for you. And thanks so very much for sharing your own experience, which is great and would definitely help us improve the protocol as provided on our website. We hope to provide more high-quality author-validated protocols to share among our research community.
I believe that you have taken right action for PCR, by using modified primer, touchdown strategy and DMSO, to improve amplification specificity/efficacy in your case.
Most Taq enzymes, unless otherwise modified, do not possess 3'->5' proof-reading exonuclease activities. Please check the info that I have found from NEB
http://www.neb.com/nebecomm/products/faqproductm0273.asp
In my case of daf-2 genotyping, I could actually distinguish heterozygote from homozygote. I could tell about two bands for heterozygotes, with wild type and daf-2 homozygotes as controls. I also used just regular desalted primers for genotyping.
BTW, to confirm the PCR genotyping results, you can also use regular Taq to amplify a PCR product (e.g., 500-bp or so) around the mutation, and then sequence the product with a primer.
Best,
Peichuan
Very glad that it worked out well for you. And thanks so very much for sharing your own experience, which is great and would definitely help us improve the protocol as provided on our website. We hope to provide more high-quality author-validated protocols to share among our research community.
I believe that you have taken right action for PCR, by using modified primer, touchdown strategy and DMSO, to improve amplification specificity/efficacy in your case.
Most Taq enzymes, unless otherwise modified, do not possess 3'->5' proof-reading exonuclease activities. Please check the info that I have found from NEB
http://www.neb.com/nebecomm/products/faqproductm0273.asp
In my case of daf-2 genotyping, I could actually distinguish heterozygote from homozygote. I could tell about two bands for heterozygotes, with wild type and daf-2 homozygotes as controls. I also used just regular desalted primers for genotyping.
BTW, to confirm the PCR genotyping results, you can also use regular Taq to amplify a PCR product (e.g., 500-bp or so) around the mutation, and then sequence the product with a primer.
Best,
Peichuan
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