HPLC purification of 454 fusion primers

[skolyar]

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]

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]

Thanks Martinjf!