HPLC purification of 454 fusion primers

skolyar · 09-20-2010, 08:10 PM

Hi there,

I am planning to use deep amplicon sequencing using 454 GS FLX Titanium series. The amplicons will be produced using conventional PCR with MID-labeled fusion primers.

Because of the large number of samples to be pooled and analysed, this experiment will involve about 100 of MID-labeled fusion primers. The cost of HPLC purification for so many primers is quite substantial.

How important is it to use HPLC purified compared to standard desalted primers in the initial PCR, provided that the PCR products will be subsequently size separated on a gel and purified using AMPure beads?

Has anyone got any suggestions?

Many thanks

martinjf · 09-22-2010, 05:20 AM

In our experience it is not important. We did not purify the fusion primers in previous experiments (this makes a total of around 2 500 primers distributed in 5 large projects). We did not detect any loss for a specific combination.

Good luck with your project !

skolyar · 09-22-2010, 01:40 PM

Thanks Martinjf!

Similar Threads
Thread	Thread Starter	Forum	Replies	Last Post
problems with M13 universal tailed fusion PCR for 454 pyrosequencing	melcar	454 Pyrosequencing	8	08-11-2013 03:44 PM
RNA-Seq: TopHat-Fusion: an algorithm for discovery of novel fusion transcripts.	Newsbot!	Literature Watch	5	07-13-2013 01:02 AM
poor amplification with fusion primers	Seqasaurus	454 Pyrosequencing	3	05-23-2013 07:20 AM
454 Titanium 16S HMP primers?	cliffbeall	454 Pyrosequencing	9	05-11-2012 06:52 AM
tophat fusion --fusion-min-dist	MerFer	Bioinformatics	1	07-24-2011 08:09 PM

09-20-2010, 08:10 PM	#1
skolyar Junior Member Location: Oceania Join Date: Jul 2010 Posts: 4	HPLC purification of 454 fusion primers Hi there, I am planning to use deep amplicon sequencing using 454 GS FLX Titanium series. The amplicons will be produced using conventional PCR with MID-labeled fusion primers. Because of the large number of samples to be pooled and analysed, this experiment will involve about 100 of MID-labeled fusion primers. The cost of HPLC purification for so many primers is quite substantial. How important is it to use HPLC purified compared to standard desalted primers in the initial PCR, provided that the PCR products will be subsequently size separated on a gel and purified using AMPure beads? Has anyone got any suggestions? Many thanks

09-22-2010, 05:20 AM	#2
martinjf Member Location: Montpellier, France Join Date: Dec 2009 Posts: 14	In our experience it is not important. We did not purify the fusion primers in previous experiments (this makes a total of around 2 500 primers distributed in 5 large projects). We did not detect any loss for a specific combination. Good luck with your project !

09-22-2010, 01:40 PM	#3
skolyar Junior Member Location: Oceania Join Date: Jul 2010 Posts: 4	Thanks Martinjf!