what primer purification level is needed?

[greigite]

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?

[SperSeq]

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?
Thanks!

[ZAAB]

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.

[protist]

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?
Thanks!

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.

[Akira]

Desalting is really fine for both custom adapters and custom primers? HPLC is quite expensive.

[ZAAB]

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.


Z

[Akira]

Thanks for the reply. How about custom oligos for adapters? Desalted is sufficient enough?

[protist]

For the sequencing primers/amplification primers I have used non HPLC purified variants (they worked fine) but for the adapters we always HPLC purify.

[ZAAB]

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.

[Akira]

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)