Issue with recent change to capture beads and amplification primer in new emPCR lots - Page 2

bradfish · 08-08-2012, 03:59 PM

I've been loading one emPCR at a time as I figured letting it sit would be an issue. I usually pause when you add the amplicon to the oil, and just move forward with one from there all the way through the procedure, then add amplicon to the next oil and start from there again. Seems to work great.

Interesting thoughts on diversity of pool correlating with signal interference. What I am are pools of many different samples each containing at least a few different organisms, many containing much more. I am going to look into this correlation when I get time.

boerm · 10-02-2012, 02:13 PM

If it is not the enrichment beads causing the problem and not the emPCR reagents, could the problem of short reads be caused by the MID adaptors, that form dimers during the library preparation.

Can it be that these dimers are not visible in the bioanalyser because they are sticking to the larger DNA fragments?

Our MID adaptor kit is over the expire date, could this explain the problem?

DNA_Dan · 10-22-2012, 01:49 PM

Quote:

Originally Posted by boerm

If it is not the enrichment beads causing the problem and not the emPCR reagents, could the problem of short reads be caused by the MID adaptors, that form dimers during the library preparation.

Can it be that these dimers are not visible in the bioanalyser because they are sticking to the larger DNA fragments?

Our MID adaptor kit is over the expire date, could this explain the problem?

This might be a possibility, but the equilibrium favors dimer formation. If you want to test for this simply do an amplification on the final library and run it on a BA chip again. If those fragments are there you should pick them up.

YMMV on expired reagents. It just depends how precious your samples are. Other labs with unlimited budgets throw them out.

boerm · 10-23-2012, 01:52 AM

Quote:

Originally Posted by DNA_Dan

This might be a possibility, but the equilibrium favors dimer formation. If you want to test for this simply do an amplification on the final library and run it on a BA chip again. If those fragments are there you should pick them up.

YMMV on expired reagents. It just depends how precious your samples are. Other labs with unlimited budgets throw them out.

Dear DNA_DAN,

Thanks for your suggestions, do you have the sequence of the Lib-L primers to amplify this library?

Thanks,

Martin.

DNA_Dan · 10-23-2012, 11:10 AM

See Application Brief # 001-2009 Entitled Unidirectional Sequencing of Amplicon Libraries Using the GS FLX TItanium emPCR Kits. (Lib-L) On the second page, Primer A and Primer B are for fusion primers. Just order these sequences up to (not including) the key pass sequence.

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08-08-2012, 03:59 PM	#21
bradfish Member Location: Colorado Join Date: Jul 2012 Posts: 20	I've been loading one emPCR at a time as I figured letting it sit would be an issue. I usually pause when you add the amplicon to the oil, and just move forward with one from there all the way through the procedure, then add amplicon to the next oil and start from there again. Seems to work great. Interesting thoughts on diversity of pool correlating with signal interference. What I am are pools of many different samples each containing at least a few different organisms, many containing much more. I am going to look into this correlation when I get time.

10-02-2012, 02:13 PM	#22
boerm Junior Member Location: Amsterdam Join Date: Sep 2012 Posts: 5	If it is not the enrichment beads causing the problem and not the emPCR reagents, could the problem of short reads be caused by the MID adaptors, that form dimers during the library preparation. Can it be that these dimers are not visible in the bioanalyser because they are sticking to the larger DNA fragments? Our MID adaptor kit is over the expire date, could this explain the problem?

10-23-2012, 11:10 AM	#25
DNA_Dan Member Location: Montana Join Date: Nov 2008 Posts: 21	See Application Brief # 001-2009 Entitled Unidirectional Sequencing of Amplicon Libraries Using the GS FLX TItanium emPCR Kits. (Lib-L) On the second page, Primer A and Primer B are for fusion primers. Just order these sequences up to (not including) the key pass sequence.