Dehydrating and Reconstituting primers - (Jun/10/2014 )
I have a batch of new primers that was reconstituted incorrectly (wrong volume of water was added to make the concentration 100uM)
I wanted to know if I can dehydrate these samples completely and then try reconstituting them once again with the proper volume.
Has anybody tried this? Has it worked for anyone?
Please let me know.
Thanks,
Ameya
it should work with a speedvac (or equivalent).
how much off were you? couldn't you just recalculate the volume required for your reactions (is there room in your reactions?)?
Actually, Its a batch of primers that our lab orders quite regularly. Usually, we add about 150ul of water for reconsitution but then the technical document along with the oligo order said that 511ul should be added to vial, which our technician followed to the word, and the PCRs stopped working.
I also wanted to know why would yield vary during separate batches of oligonucleotide synthesis.
If I understand correctly, you generally add 150uL to your lyophilized primers to make a concentration of 100uM, but this time your technician added 511uL to the lyophilized primers? If so, this will give you a concentration of about 30uM. Since you add ~200-400nM primers to your PCR, just dilute your stock (30uM) accordingly.
Ahrenhase,
Thank you for your suggestion. mdfenko, too suggested the same and I should probably try that out.
But the more I look at technical sheet that came along with previous oligo orders, the more I begin to doubt this set of primers that I have received.
There is a significant difference in the yield during synthesis and accordingly the data sheet specified larger volume of water to be added for reconstitution.
Would anybody know why this could happen?
they may have purchased a new, more efficient synthesizer? maybe they cleaned the synthesizer or used fresh chemistry? maybe the tech who made this batch was better?
it's also possible that they didn't clean it up as well so it appears to have more but really has the same amount or less of the specific nucleotide.
have you determined the concentration with a spectrophotometer?
Remarkably, the weather has a lot to do with it. Synthesis reagents are very moisture sensitive, and high humidity dramatically reduces yield of full length product.
mdfenko on Fri Jun 13 15:15:44 2014 said:
they may have purchased a new, more efficient synthesizer? maybe they cleaned the synthesizer or used fresh chemistry? maybe the tech who made this batch was better?
it's also possible that they didn't clean it up as well so it appears to have more but really has the same amount or less of the specific nucleotide.
have you determined the concentration with a spectrophotometer?
phage434 on Fri Jun 13 17:06:34 2014 said:
Remarkably, the weather has a lot to do with it. Synthesis reagents are very moisture sensitive, and high humidity dramatically reduces yield of full length product.
Thanks for your answers I haven't determined the concentration with a spec, but am quite surprised that such little environmental changes can have significantly high impact on the oligo synthesis.
How do you guys ensure that the primer batch you are getting is unaffected by all this. And doesn't this make reproducing results from another far away lab all the even more difficult?
order them cleaned by hplc or page
Trityl measurements during synthesis give a good estimate of yield of each step of the synthesis. That, along with spectroscopic measurements of the final DNA concentration, provide good information about amounts of the final product. For the most part, the yield of oligos is of very little importance. They are usually used in significant excess is both PCR and sequencing reactions. Possibly in situations where they are being duplexed to make dsDNA, the relative amounts may matter.