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Old 10-26-2011, 05:50 PM   #1
skblazer
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Default Is the PCR purification step between ligation and size selection necessary?

Hello,

This is my first time to construct an Illumina Paired-end library. According to the protocols, there is a PCR purification step after ligation of adapters and prior to size selection step. If I want to perform the gel size-selection before PCR enrichment, is this PCR purification step necessary?

I guess I can directly run the ligation product on a gel for size selection, but both Illumina native kit and NEBNext kit include that PCR purification step?

Best wishes,
SK
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Old 10-27-2011, 08:55 AM   #2
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I don't see any reason why it would be necessary or useful.
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Old 10-27-2011, 09:41 AM   #3
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The PCR insures that your library inserts are flanked by adapters, a prerequisite for cluster formation and sequencing, and also increases the amount of library. PCR-free protocols have been used successfully, but you should quantify your library by qPCR so that only adapter-ligated molecules are measured.
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Old 10-27-2011, 10:57 AM   #4
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Sorry, I didn't mean the PCR step. I mentioned a QIAquick PCR purification step after ligation but before size selection on a gel.

For example, the last step of "Adaptor ligation of dA-Tailed DNA" is "Purify DNA sample on one column and elute in 30uL of sterile dH2) or elution buffer" both in NEBNext kit and Illumina Paired End Library kit.

I want to know if I can directly add ligation product to a 2% gel for size selection without this purification step.
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Originally Posted by HESmith View Post
The PCR insures that your library inserts are flanked by adapters, a prerequisite for cluster formation and sequencing, and also increases the amount of library. PCR-free protocols have been used successfully, but you should quantify your library by qPCR so that only adapter-ligated molecules are measured.
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Old 10-27-2011, 10:58 AM   #5
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Quote:
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I don't see any reason why it would be necessary or useful.
All right, I'm just wondering I missed some critical steps that I don't understand.

Thanks for the advice
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Old 10-27-2011, 12:04 PM   #6
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In principle, the amounts of primers and other small stuff in a PCR reaction might overwhelm the capacity of some gels. Some tiny amount of small material might end up co-migrating with the DNA you intend to cut out.

More likely, the PCR reactants will slightly alter the mobility of your DNA, making it not run at the same rate as your ladder. So your size estimate may be off. I guess you could add 10x PCR reaction mix (withOUT polymerase!) to your ladder to give it the same buffer and ionic strength as your PCR reaction...

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Last edited by pmiguel; 10-28-2011 at 03:41 AM. Reason: You probably do not want to add polymerase to your ladder!
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Old 10-27-2011, 06:15 PM   #7
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Thanks Phillip. The reaction mix is super expensive, thus I think I still need this PCR purification.
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Originally Posted by pmiguel View Post
In principle, the amounts of primers and other small stuff in a PCR reaction might overwhelm the capacity of some gels. Some tiny amount of small material might end up co-migrating with the DNA you intend to cut out.

More likely, the PCR reactants will slightly alter the mobility of your DNA, making it not run at the same rate as your ladder. So your size estimate may be off. I guess you could add 10x PCR reaction mix (with polymerase!) to your ladder to give it the same buffer and ionic strength as your PCR reaction...

--
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Old 10-28-2011, 03:44 AM   #8
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Sorry, just to be clear--don't add polymerase to your ladder (typo in my initial post that I just corrected)!

Also, the size shift would likely be minor under most circumstances.

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