hello everyone
i am trying to set a pcr in my lab and now i dont know how to made an 10x ammonium buffer.i searched a lot but i couldnt find any protocol for this. i dont know the concentration of tris or ammonium sulfat that i should use in this buffer
i will be happy if anyone help me
Tris? Ammonium? Those have different pKa values. Tris is around 8.3 (pKa) while ammonia is around 9.3. You need to first specify the pH of the buffer desired.
Never heard of needing Ammonium sulfate for PCR.
PCR needs the following components: template DNA, MgCl2, dNTPs, oligonucleotide primers, and a thermostable DNA polymerase such as Phusion High Fidelity DNA polymerase. Use of 3% (v/v) DMSO final concentration usually makes PCR work better, in my experience.
Here is a basic PCR protocol:
https://www.neb.com/protocols/0001/01/01/pcr-protocol-m0530
Not sure what the composition of 5X Phusion HF Buffer is but I think it is probably 5 * {25 mM TAPS-HCl (pH 9.3 @ 25°C), 50 mM KCl, 2 mM MgCl2, 1 mM b-mercaptoethanol}, based on the information from New England Biolabs. You can buy it.
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