Required oligo purity for homemade library preps?

zaratieg · 10-18-2013, 06:48 AM

Hi, we are gearing up to make some home-made libraries with custom oligos. I have never ordered oligos for library construction, what are the purity requirements? HPLC, PAGE purification? Is there a difference between the ligation adapters and the PCR primers in terms of purity? Will desalted primers be OK for PCR?

mcnelson.phd · 10-18-2013, 11:44 AM

The best answer is that if you're having primers synthesized for Next Gen sequencing, regardless of platform, then they should be HPLC purified or better. The reason is that primers are synthesized 3' -> 5', so if the 5' end is missing a base or two then that can negatively affect sequencing.

Stating that, I know a lot of people who have ordered primers for amplicon libraries without HPLC purification, and they haven't had significant issues. In some ways it comes down to how tolerant are you to failure; HPLC purification will generally guarantee you that your primers are good while the extra cost may not be worth it if you're fine with having some samples run poorly on occasion.

zaratieg · 10-18-2013, 12:05 PM

Thanks! My tolerance of failure is dwindling in parallel to my startup money, so I'll spend the extra bucks.

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10-18-2013, 06:48 AM	#1
zaratieg Member Location: NYC Join Date: Jun 2010 Posts: 18	Required oligo purity for homemade library preps? Hi, we are gearing up to make some home-made libraries with custom oligos. I have never ordered oligos for library construction, what are the purity requirements? HPLC, PAGE purification? Is there a difference between the ligation adapters and the PCR primers in terms of purity? Will desalted primers be OK for PCR?

10-18-2013, 11:44 AM	#2
mcnelson.phd Senior Member Location: Connecticut Join Date: Jul 2011 Posts: 162	The best answer is that if you're having primers synthesized for Next Gen sequencing, regardless of platform, then they should be HPLC purified or better. The reason is that primers are synthesized 3' -> 5', so if the 5' end is missing a base or two then that can negatively affect sequencing. Stating that, I know a lot of people who have ordered primers for amplicon libraries without HPLC purification, and they haven't had significant issues. In some ways it comes down to how tolerant are you to failure; HPLC purification will generally guarantee you that your primers are good while the extra cost may not be worth it if you're fine with having some samples run poorly on occasion.

10-18-2013, 12:05 PM	#3
zaratieg Member Location: NYC Join Date: Jun 2010 Posts: 18	Thanks! My tolerance of failure is dwindling in parallel to my startup money, so I'll spend the extra bucks.