I have been failing to get amplification of PCR and decided to add different concentrations of MgCl2 to my mastermix and the prepared different PCR tubes with their negative controls. The first well is the 1kb ladder, followed by 4.5mM MgCl2 mastermix with template, then the negative control. Well 4 is the 5.0mM MgCl2 mastermix with template, followed by its negative control. Well 6 is the 5.5mM MgCl2 mastermix with template, followed by its negative control and well 8 is the 6.0mM mastermix with template, followed by its negative control.
I used a P21 primer which would amplify at 206 bp but I see so many bands. The lowest band on the ladder is 1000bp.
You are adding too much Mg. Divalent ions like Mg decrease the annealing temperature of primers so they will anneal at many positions and generate non specific amplimer bands The bent running of the samples in agarose gel is typical of too much salt in the sample or possibly the voltage is too high and the gel is warming up and running badly. You will get better results by using 1.5mM magnesium and running a temperature gradient of annealing temperatures from 5c below the primer Tm to 4c above the primer Tm. The lower temperatures may be dirty and the highest temperature may fail to amplify anything but somewhere in between there will be a temperature that mainly amplifies just the one specific band that you want
In addition to Paul's comprehensive answer: Esp. when using Taq Polymerase, 200 bp are amplified in a few seconds.
Optimize the extension time. A few seconds should be enough; Even consider just to cycle between melting and annealing. The ramping time between both temperatures could be enough to amplify the desired. fragment.
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