Again while doing the PCR amplification of snail DNA, I got stuck with no visible bands on gel. But there is the presence of primer dimers in the gel. The DNA pellet I got readily dissolved in double distilled water and it is about 100ng/ul in quantity. I had added about 8ul of DNA templte to 50ul reaction mixture. What could be the possible reason of no band formation? Is it due to more amount of template DNA? or due to any of the PCR components? Does Primer Dimer formation is due to defective PCR reagents?
But primer dimers are seen on every gel.
Please help.
Hi Keerthy
You are using almost 4 times the maximum amount of genomic DNA suggested in a 50ul PCR reaction. Try reducing it to 50-250ng per reaction. Also standardize your PCR reaction for suitable annealing using a gradient.
Good luck
Pranay,
Thank you so much. I will try doing it and update my results
Hi,
As Pranay told you, try to optimize your reaction (annealing temperature, primer concentration and Magnesium concentration). Once you have your PCR optimized, don't add too much quantity of your DNA to the PCR (it's better work with 10 to 70 ng of DNA in the reaction) so, if you're adding 4uL of DNA with a concentration of 100ng/uL and the final volume of your Rx is 50 uL you're working with 80 ng in the Rx which could inhibit the reaction. Furthermore to the concentration of your primers which could be the reason of your primer dimers, analyze your set of primers with oligoanalyzer:
https://www.idtdna.com/calc/analyzer
With that software, you can introduce the conditions of your PCR such as primer concentrations, Mg, dNTPs and Na concentration so that you can get the dG of your primers in order to know if they are forming secondary structures and also if they are forming primer dimers with themselves (self dimer) or with the other primer (hetero dimer). For both (self and heterodimer) it's better to have values between -9 and 9, any dG greater than that is telling you that you have a bad set of primers and it's very probable that they're forming primer dimers due to the fact that they are complementary with each other.
Besides, you could try using Hot Start Enzyme and also works with chilled ice while you're preparing your PCR. Also, you can previously set up your machine to prewarm the lid and block of the thermocycler as well in order to have your block at 95°C (or any temperature for initial denaturation) at the time that you put the tubes in the block.
The primer dimer formation at the end of the gel is usually caused by high volumes of the primers. What happens here is that your forward and reverse primers anneal with eaach other since there are "extras". You could reduce the primer volume.
However, primer dimers may be removed after purification. And it can still be sent for sequencing despite the dimer formation (given that it is purified). Keep me posted!
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