Hi,
Please anyone already prepared the mix for Standard PCR? But without using a commercial kit. It is for staphylococcal enterotoxins genes. I am looking for the concentrations of each constituent.
Thanks
Dear Hachemi,
there are many sources of detailed info how to set up standard PCR. These are just few examples:
https://www.sigmaaldrich.com/technical-documents/protocols/biology/standard-pcr.html
https://www.bosterbio.com/protocol-and-troubleshooting/molecular-biology-protocol-pcr
http://cshprotocols.cshlp.org/content/2018/5/pdb.prot095117.full
Good luck!
I run 50 ul reactions, usually. You need to add the polymerase last, I recommend the hot start version of phusion or Q5. Use approx 35 ul MGBW, 5 ml HF buffer, 5 ul high gc buffer, 0.5 ul dmso, 0.5 ul 2um MgCl, 1 ul dntp, variable template from 5 ng plasmid to 25 ng for genomic dna, 25 ng of both forward and reverse primer for your template, 0.6 ul phusion polymerase. Avoid onetaq, it is error prone and adds an adenine to the 3', it also has twice the extension time of proof reading polymerases.
Hello,
In our lab we use taq-polymerase for standart PCR. In 50 ul reaction we use:
- 5 ul of 10x Taq Buffer
- 4ul of 2.5uM dNTPs
- 0.6 ul of 20uM primer (for each primer)
- 0.5 ul of Taq-polymerase (C=5U/ul)
- water to 50 ul
- 3-8ul of matrix depending on concentration.
Arina : your recipe is contrary to the recommendations of NEB ie you need 10 ul of buffer and almost never any extra MgCL2, but the full 10 ul of buffer would supply that magnesium
Amy, you was right. My mistake - sometimes we use our manually prepared buffer w/o MgCl2 and add extra MgCl2. Or we can use special Taq buffer (with 2,5uM MgCl2) without additional magnesium. Both variations mixed in my thoughts.
Thank you for your comment, I corrected the recommendation.
I have read all the responses. The information given is very useful. Thanks for all of you, and special thanks for you Amy Johnson
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