I've noticed when thawed my 100 µM primer stock has precipitation that can't be resuspended with vortexing or slight heating. These primers also seem to give mutant plasmids with the WT sequence at the location that was supposed to be mutated and addition of an extra nucleotide at positions throughout the primer region. It seems like the primers are quite impure, but is it worth it to brute force solubilize them and PAGE purify? Any idea what is causing the precipitiation of the oligos?
Hello Xander,
For sure the best thing you can do is to reorder your primers with a small amount of dollars. I would not work in these conditions.. first of all beacuse if those are not primes for mutagenesis it's means that there is a contamination, second because your stock should be oligo plus water so I don't know what could be your insoluble precipitation.
Good luck
Edoardo
Hi there,
Oligos are very soluble at 100µM therefore the insoluble you see is either salt or residual resin from cartridge purification. But I don't think this is the reason why your mutagenesis is not working properly. What was the quality you asked for when ordering? To get sure about the sequence HPLC purification and MS analyses are required.
Hi Dominique,
Thanks for the feedback. I just did regular desalting on these primers, but I'm going to reorder them with higher quality (optimally HPLC depending on the cost, but definitely PAGE purified). It seems like all of the mistakes in the sequence are in the primer region so I'm fairly confident it's an issue with the purity of the primers.
Double-check your primer sequences before you order them. It's easy to make a silly mistake, especilly if you type them in by hand. I'd put money on either bad chromatogram sequences or ordering the wrong primers, not a mysterious "mutation process" due to salt-purified primers.
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