I had added MgCl2 buffer twice in PCR master mixture & unable to find any PCR Product.
I think it is good that you first understand the role of magnesium chloride in a PCR reaction which is to act as a cofactor/catalyst to the thermostable polymerase. too much or too little of it can affect the reaction. If it is too much, the polymerase will act too quickly and can cause errors in adding the nucleotides and if it is too little then the reaction will be slowed down and you will not obtain adequate amplicon. so you might consider it as a possibility for not getting the product assuming that everything else was done right. all the best
Magnesium chloride in PCR:
An essential cofactor for the DNA polymerase in PCR is Magnesium chloride. Its concentration must be optimized for every primer : template system. Many components of the reaction bind magnesium ion, including primers, template, PCR products and dNTPs. Because it is necessary for free magnesium ion to serve as an enzyme cofactor in PCR, the total magnesium ion concentration must exceed the total dNTP concentration.
During replication dntp's gets broken down to dnmp's to form phosphodiester bond between 3' OH of adjacent nucleotide and 5' Phosphate of the upcoming nucleotide. in this reaction Mg ion binds to the alpha phosphate group of dntp and helps in the removal of beta and gamma Phosphate from dntp.
Increasing Mg ion enhances Taq activity but at the expense of specificity, whereas less Mg ion lowers Taq's activity but increases its specificity.
Further Mg ion increases the Tm of two interacting DNA strands by shielding the negatively charged phosphodiester backbone and decreasing the electrostatic repulsion between DNA strands.
Dear Dr. Arthur I am sure that Mr. Sudheer is a novice in both english literature and research and he does not intend to be rude. Its just about putting up with him and trying to help him learn, if possible. He is a student here.
I do agree with you that the way this question asked is inappropriate.
Cheers!
Dear Mr. Sudheer its my kind request to have your senior or a well wisher in the Institute to demonstrate setting up PCR to you at least couple of times.
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