Hello everyone,
I'm very confused right now.
I have a pair of primers that works fine with DreamTaq polymerase. I see the fragment in the gel and it is of correct size.
However, when I tried to amplify the same fragment with Q5, it just did not work. And Phusion polymerase did not work as well.
Do you maybe have any ideas why that happens and how to overcome it?
Best regards,
Nataliya
Some of the higher quality polymerases are very fussy about the amount of free magnesium in the mix. DNA,ntps and edta all sequester Mg and this may be making a difference to the enzyme working. It may be worth titrating the reaction at 1.5, 2.0 and 2.5 mM Mg if you are getting no amplification.
Hi Nataliya,
Please refer to the following article:
https://www.nature.com/articles/srep28769
According to the above-mentioned study, oligonucleotides containing G-rich sequences such as GGGGG and GGGGHGG can inhibit proofreading DNA polymerases in a dose-dependent manner. So, please do check your primer sequences and if you should change their concentrations.
Good luck
Hello Nataliya,
Usually when using new polymerases its recommended to test the optimal amount of Mg for the reaction. Start with 1mM up till 2.5mM for rxn.
Hello everyone,
thank you all for the valuable advice. Unfortunately, I am still not able to produce the fragment, so I will just redesign the primers.
Best,
Nataliya
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