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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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.
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Originally posted by SperSeq View PostHi,
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!
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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.
Z
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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.
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