I need to run PCR using various published primers. For one of the primers I intend to use BJ1 & BN2. For this primer a paper reported using 2.5ul of 10x PCR buffer containing 15mM MgCl2 while another paper that used the same primer was silent on MgCl2 . For another primer I will be using, Hep 300 & Hep 900, a paper reported 1.0mM MgCl2 in her reaction mixture. For a third primer HaemF and HaemR2, a paper reported using 1.5mM MgCl2. Since the MgCl2 concentration reportedly use for each of these primers is different, is it the primers that determines the concentration of the MgCl2 to be used?
To run my PCR I intend buying a ready to load PCR Master Mix which comes in 7.5mM MgCl2 and 12.5mM MgCl2. I will have to decide which to go for of the two. During my PCR will I have to be diluting the PCR Master Mix differently for each of these primers for good results base on the published conditions?
You should just test different MgCl concentrations and see which one works best with your primer pair. What is optimal concentration depends on what enzyme you use (its a cofactor for the enzyme) and the concentration of dNTP (they bind each other, thus affect each other). There are rules of thumb for best MgCl concentrations, but in the end, when establishing a new PCR using your particular reactionmix, enzyme, PCR machine etc you should just test it out.
hope this helps!
I think that you should buy separately polimerase, nucleotides, MgCl2. In this case you can add MgCl2 in different voulme to your reaction (its depends on kind of primers). Preparing a master mix of these reagents does not take much time and you are sure that MgCl2 concentration is correct.
Diluting will not help. If you decided buy ready to use MASTER MIX you should order MgCl2 as well. In this case you should prepare your samples and add to each different of MgCl2 volumes (you remember that your MASTER MIX consist certain concentration of MgCl2 so You need to calculate how much extra MgCl2 you need to add to the master mix, which contains MgCl2).
:)
Magnesium ions bind in the major groove between phosphate groups and significantly alter DNA/RNA stability. Most PCR kits furnish a buffer that provides approximately 1.5 mM Mg2+, many studies have shown that optimizing Mg2+ can significantly enhance or diminish PCR efficiency. I would say your probably safe in the range of 1.5-5 mM Mg2+, any more than that and you risk throwing your Tm to far off the theoretical causing annealing issues, any less and the instability of your DNA backbone will cause issues during polymerization.
the rule for old polymerases used to be that the enzyme needed 1mM Mg as a cofactor to be active. Unfortunately for pcr many things remove Mg like DNA. NTPs EDTA,primers and impurities in the dna ( especially in soil dna) so you have to add more MG so that when these inhibitors have removed MG there is still 1mM left for the enzyme. In practice as suggested above this may mean titrating Mg concentrations to see what works best for your primer sets and dna and is often 1,5 -3 mM. Some modern polymerases for long pcr are especially fussy about Mg concentrations and too much MG ( 4mM and above) will cause more non specific bands to be produced. When setting up primers use too much MG ( 2.5mM) to ensure getting amplification even if it is a bit dirty then reduce the Mg to 1.5 mM for higher specificity once it is working. If using master mixes the company has done all the work needed in setup and just follow their recommendation
This depends on the polymerae you use. See specification of the supplier!
1. A lot of PCR first-time doers can be confused by the 'original concentration' and 'final concentration' of a PCR compoent appeared on the instruction sheet. For example, you mentioned 3 different MgCl2 concentrations (15mM, 1.5mM and 1.0mM) appeared in 3 different papers in your question. However, if you look carefully at the first one "using 2.5ul of 10x PCR buffer containing 15mM MgCl2.....", this is just the original concentration of MgCl2. If you prepare a PCR with 25 uL final reaction volume and use '2.5ul of 10x PCR buffer containing 15mM MgCl2', then you still get a final MgCl2 concentration at (2.5 uL)/(25 uL) x 15mM= 1.5mM. Not much different from the other two concentrations mentioned in the other two papers.
2. If MgCl2 is not enough in a PCR reaction, it can reduce the yield of PCR product. If too much, unspecific PCR bands can be amplified (see attachment). In the attachment, it seems that MgCl2 with 1.5mM~2mM gave good PCR results**.
**Source: from FisherThermoTM Scientific [https://www.thermofisher.com/us/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/pcr-education/pcr-reagents-enzymes/pcr-component-considerations.html]
3. NEB suggestes using 1.5-2mM (final concentration) of MgCl2 in general PCR
I suggest try using different concentrations of MgCl2 in your reaction mix. Years ago (1996), I have optimized an inverse PCR method using a PCR Optimizer kit purchased from Invitrogen. I am hoping that this kit containing different MgCl2 concentrations is still available. Please check my publication on this optimization procedure (entitled, "Optimization of Inverse PCR) that I just uploaded to my profile, and also attached here. Hoping that this will be of some help. All the best.
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