SequalPrep normalization plates and low amount of starting material

[meri.lindqvist]

Hi,

I have been looking into Invitrogen's (ThermoFisher Scientific) SequalPrep normalization plates for a quick way to clean and normalize barcoded DNA-libraries (amplicons) for IonTorrent sequencing. The protocol looks easy and the price is not too bad, so I'd love to use this method instead of our current individual Qubit measurements and pooling which works fine but is quite time consuming.

My main concern is with the amount of starting material that is required in the protocol, "at least 250 ng PCR product (amplicon)". I don't think I have this much DNA after my PCR which is low in volume and has a lowish number of cycles. Has anyone else used the SequalPrep kit with lower DNA amounts than specified in the protocol? Would it still work if starting with less than 250 ng??

Cheers,
Meri

[ljmullen2]

I've run into this issue as well. It seems that with <250ng starting material, the kit sometime still works (still produces at least some reads for that sample), but definitely not guaranteed to produce as many reads as a sample with >250ng starting, and may not work at all. Whenever I have this issue I incubate overnight at the binding step and vortex multiple times during the incubation, all to increase the chances of the few amplicons in the well finding the binding sites.