when i do PCR, band show me always smear.
I add more Mg2Cl so Smear has been removed to some extent.
but band brightness is decrease.
i attached img file. this is not exact samples but look like this.
As i know, The more magnesium you add, the higher specific the band's concentration.
sorry my poor English
Too low/high conc. of Mg, are both bad for PCR reactions. High Mg conc. stabilize the double stranded DNA, so there's chance of partial denaturation and erroneous amplification. High Mg also stabilize primer binding and improve the chances of non-specific annealing.
Not sure what exactly happened in your case. May be, instead of increasing Mg rather decrease the conc. a bit, also play with other parameters (annealing temp., enzyme /primer conc. etc.)
Too low/high conc. of Mg, are both bad for PCR reactions. High Mg conc. stabilize the double stranded DNA, so there's chance of partial denaturation and erroneous amplification. High Mg also stabilize primer binding and improve the chances of non-specific annealing.
Not sure what exactly happened in your case. May be, instead of increasing Mg rather decrease the conc. a bit, also play with other parameters (annealing temp., enzyme /primer conc. etc.)
Increasing magnesium lowers the annealing temperature of primers so will make a good pcr less specific. If the template dna is poor quality and contains pcr inhibitors then more Mg may make the pcr work better. Without a no template control or size ladder it is not clear to me that the top gel shows amplification or just too much degraded dna and a primer dimer in which case the lower gel is working better not worse but it would be useful to know how much dna is used in each reaction and the purity of that dna
[Mg] has to be optimized, with higher [Mg] (within a range recommended) you increase processivity of your polymerase in PCR BUT the specificity suffers and as a result you may/will see smears or/and unspecific bands. To what extent higherMg impacts loss of specificity depends on your primers/temperature/PCR conditions/your locus of interest (e.g., high or low in G/C or/and 2ndary structure). When this drop in specificity happens, the enzyme is amplifying a bunch of other loci (you may not see it as a band but as smear = many different bands). This means that your enzyme is not focusing entirely on amplifying your locus of interest. This means that you will see drop in PCR yield of your locus you're trying to amplify. You can shorten amplification time (68 or 72C step, depending on the enzyme), this usually reduces smear by not allowing longer unspecific loci to be amplified. Hope it helps, seems like "blast from the past" question
Higher Mg will make the Taq polymerase more processive and this will increase the yield of the PCR product, however, the PCR reaction becomes less specific (due to lowering of the annealing temperature of the primers). So, adding Mg to PCR will enhance the yield of the reaction up to an optimal Mg concentration. Above the optimal Mg concentration, the specificity of the PCR reaction will decrease and non-specific PCR products will appear and may cause smear appearance. A solution to the smear problem is to cut the specific PCR product band and do a gel purification.
All people helped me. Thank you for yours answer. i will try to follow answers.
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