Fluidigm PCR amplicon Library prep

yog77 · 06-06-2012, 09:01 AM

Hi has any one got any advice on a two-step PCR amplicon library generation via barcode/adapter addition using a secondary PCR.

I am using the Fluidigm AA system have generated my Primary PCR amplicons 48 samples each with 48 amplocons and now need to attach the barcode/adapter via a secondary PCR. Fluidigm suggest using a 1in100 dilution of the primary PCR and adding the adapters using 15 cycles in a 20uL PCR.

My question is would it not be better to use a less dilute amount of the primary PCR say 1in20 or 1in40 dilution and cut the number of cycles to 12 or 13 and even in half the reaction volume (say 10uL), therefore reducing PCR errors by using fewer additional cycles? I just wondered if these modifications would be beneficial?

Thanks

ugm6hr · 10-05-2012, 03:57 AM

Quote:

Originally Posted by yog77

Hi has any one got any advice on a two-step PCR amplicon library generation via barcode/adapter addition using a secondary PCR.

I am using the Fluidigm AA system have generated my Primary PCR amplicons 48 samples each with 48 amplocons and now need to attach the barcode/adapter via a secondary PCR. Fluidigm suggest using a 1in100 dilution of the primary PCR and adding the adapters using 15 cycles in a 20uL PCR.

My question is would it not be better to use a less dilute amount of the primary PCR say 1in20 or 1in40 dilution and cut the number of cycles to 12 or 13 and even in half the reaction volume (say 10uL), therefore reducing PCR errors by using fewer additional cycles? I just wondered if these modifications would be beneficial?

Thanks

This makes perfect sense, but I didn't want to deviate from their published protocols for my experiment.
If you do use an alternate dilution / number of PCR cycles, I'd be interested to hear the outcome.

yog77 · 10-30-2012, 04:36 AM

After trying a few different dilutions and cycle combinations and checking them on the bioanalyzer. I went with the following:

1uL of the primary PCR product as template for a 5uL secondary/barcoding PCR with only 8 cycles. This worked very well and gave good sequencing results. If you require further details let me know.

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06-06-2012, 09:01 AM	#1
yog77 Member Location: London Join Date: Jun 2011 Posts: 18	Fluidigm PCR amplicon Library prep Hi has any one got any advice on a two-step PCR amplicon library generation via barcode/adapter addition using a secondary PCR. I am using the Fluidigm AA system have generated my Primary PCR amplicons 48 samples each with 48 amplocons and now need to attach the barcode/adapter via a secondary PCR. Fluidigm suggest using a 1in100 dilution of the primary PCR and adding the adapters using 15 cycles in a 20uL PCR. My question is would it not be better to use a less dilute amount of the primary PCR say 1in20 or 1in40 dilution and cut the number of cycles to 12 or 13 and even in half the reaction volume (say 10uL), therefore reducing PCR errors by using fewer additional cycles? I just wondered if these modifications would be beneficial? Thanks

10-30-2012, 04:36 AM	#3
yog77 Member Location: London Join Date: Jun 2011 Posts: 18	After trying a few different dilutions and cycle combinations and checking them on the bioanalyzer. I went with the following: 1uL of the primary PCR product as template for a 5uL secondary/barcoding PCR with only 8 cycles. This worked very well and gave good sequencing results. If you require further details let me know.