Can anyone please tell me if you tried PCR run without the need of adding an extra aliquot of MgCl2 and still have got the amplification? I tried with two suppliers but they didn’t work out for me. I usually add the same volume in a reaction mix, as it is needed for a buffer without MgCl2.
Hi,
PCR will not work without Magnesium Chloride, As Mg2+ is essential for Taq polymerase. Generally 1.5mM to 6mM of MgCl2 is used in PCR reaction.
Before adjusting MgCl2, I would recommend you optimize your primer annealing temparature keeping MgCl2 concentration constant in the above specified range.
Before setting up next PCR check these:
1. Verifying primer / probe concentration of your stock (you can check OD260)
2. Include Positive and Negative controls
3. Verify your reagents are not expired.
4. Quantify your input sample (DNA/RNA)
I concur with the previous answers. I definitely prefer adding my own Mg2+ to a magnesium-free 10x reaction buffer after diluting it to make my PCR mix. For PCR with Taq, I usually start with 2.0 mM MgCl2.
Note that certain DNA polymerases e.g. the proof-reading Pfu polymerase prefer MgSO4 to MgCl2.
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