what primer purification level is needed?
I'd like to order some custom oligos for multiplexing using the Illumina adapter/primer sequences, but am not sure what purification level to get (desalt or HPLC). Any experiences/recommendations with homemade primers? Do the deletion/truncation products left after desalting interfere with downstream reactions?
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same question about purification
Hi,
I have the same question. Did you have any feedback? Maybe,since 2 years have passed, you are now the expert. Do you have any recommendation from your experience?
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Late reply:
Illumina states "HPLC is what we use in the lab".
In the trenches, the guys in my core facility says desalted is fine.
Just make sure your Tm is ~74 degrees, and calculate using the Illumina primer as your comparison regardless of the program you use to calculate Tm.Comment
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We have been using 'home-brew' MWG synthesized adapters and primers since Feb 2009 and always HPLC purify. We have had no issues with our stocks.Originally posted by SperSeq View Post
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Desalting is really fine for both custom adapters and custom primers? HPLC is quite expensive.
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WE've been using standard desalted custom sequencing primers for over a year now, and have had excellent results...
Just remember to get the Tm up to ~70 degrees, so if this requires longer primers, choose a vendor that guarantees the quality of the synthesis...OR check by sequencing a PCR product, cloned into E.coli, derived from your Illumina-ready samples.
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Thanks for the reply. How about custom oligos for adapters? Desalted is sufficient enough?Comment
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For the sequencing primers/amplification primers I have used non HPLC purified variants (they worked fine) but for the adapters we always HPLC purify.Comment
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When we build our own adapters, we perform this using standard desalted primers as well.
We add about half of the illumina adapter seq in a primary PCR (we're sequencing a PCR product whose 5' and 3' ends are known and constant), and then install the ends of the illumina adapters using a second PCR step.
All of these primers are regular desalted ~40mers.Comment
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I see. I am now thinking whether to choose oligo synthesized by IDT or Sigma. Any idea which is better? (*wondering if I could ask this question)Comment
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