Gel run before AMPure

MonaE · 07-19-2013, 06:20 PM

I am using targeted sequencing designed by Qiagen. I would like to perform a gel run for my samples before measuring them by Qubid and AMPure step. The reason I want to do that is because if I depended ONLY on Qubid I will have a measurement for everything in my sample, while when I run a gel I can see my targeted amplicons based on their size in the gel. Does any one agree with me? if disagree can you tell me why and correct me if I am wrong?

Heisman · 07-19-2013, 09:05 PM

With a normal ampure cleanup you can get rid of primers. Depending on exactly what you are doing you can do some size selection as well. Typically people will use a bioanalyzer or a tapestation as a final step to QC their library and to quantify it. If you do a gel extraction first you can lose a lot of material and possibly sacrifice the complexity of your library.

MonaE · 07-20-2013, 01:45 AM

Thank you for your replay. I want to use the gel just to check the (efficiency) of my PCR that is all. I will not cut it or size select. This is becasue when I measure with quibid I get reading that is much higher than after AMPure which is a very tuff experiance for my sample

as they end up with much lower reading after the AMPure. So I do not know if this low reading becasue my PCR was not working well from the begining or a result of the AMPure step.

Heisman · 07-20-2013, 06:35 AM

Ah, if you're just troubleshooting then go for it. Then you can determine if the problem is the PCR or the Ampure cleanup.

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07-19-2013, 06:20 PM	#1
MonaE Member Location: France Join Date: May 2013 Posts: 24	Gel run before AMPure I am using targeted sequencing designed by Qiagen. I would like to perform a gel run for my samples before measuring them by Qubid and AMPure step. The reason I want to do that is because if I depended ONLY on Qubid I will have a measurement for everything in my sample, while when I run a gel I can see my targeted amplicons based on their size in the gel. Does any one agree with me? if disagree can you tell me why and correct me if I am wrong?

07-19-2013, 09:05 PM	#2
Heisman Senior Member Location: St. Louis Join Date: Dec 2010 Posts: 535	With a normal ampure cleanup you can get rid of primers. Depending on exactly what you are doing you can do some size selection as well. Typically people will use a bioanalyzer or a tapestation as a final step to QC their library and to quantify it. If you do a gel extraction first you can lose a lot of material and possibly sacrifice the complexity of your library.

07-20-2013, 01:45 AM	#3
MonaE Member Location: France Join Date: May 2013 Posts: 24	Thank you for your replay. I want to use the gel just to check the (efficiency) of my PCR that is all. I will not cut it or size select. This is becasue when I measure with quibid I get reading that is much higher than after AMPure which is a very tuff experiance for my sample as they end up with much lower reading after the AMPure. So I do not know if this low reading becasue my PCR was not working well from the begining or a result of the AMPure step.

07-20-2013, 06:35 AM	#4
Heisman Senior Member Location: St. Louis Join Date: Dec 2010 Posts: 535	Ah, if you're just troubleshooting then go for it. Then you can determine if the problem is the PCR or the Ampure cleanup.