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!
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.
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
- Badges
- Science method