Platinum Hot Start versus Q5 Hot Start Hifi PCR kits

[tarunaaggarwal]

Dear EMP protocol users - I have been using EMP's 18S protocol to sequence eDNA from marine sediments. Before ordering the expensive 18S ultra primers, we ordered just the gene specific primers for testing purposes. I run PCR with two kits using these 1391F and EukBr non-barcoded primers- Invitrogen's Platinum Hot Start 2x master mix and NEB's Q5 Hot Start Hifi 2x master mix.

I got much stronger bands with Q5 than Platinum Hot Start (see gel image). NEB claims that Q5 has much better fidelity compared to Platinum Hot Start.

So I'm thinking about switching PCR kits but wanted to check if anyone else has tried a different PCR kit with EMP protocols regarding of the gene? I would like to stick to EMP's protocol if possible but I also have concerns about their recommended PCR kit.

Any suggestions or thoughts are appreciated. Thank you! -Taruna

[cuttlefishh]

This comment from Jeremiah Minich (Knight Lab) may be helpful:

I think either way won’t make much of a difference in overall success...(switching mastermix or using the one with fair bands). From my experience, band intensity is useful but very very difficult to quantify for any sort of sample. You really need to sequence it to know especially for samples of low biomass. If the marine sediments are however chalk full of metals, it may indeed cause problems with mastermix performing poorly ... so maybe this other mastermix has some chelators to be resistant?

[tarunaaggarwal]

Thank you for responding, Luke. So I have a curious question for Jeremiah. Costello 2009 in Science is one of the original papers that EMP references mention. Costello 2009 states that they used two PCR kits Invitrogen's Platinum PCR SuperMix or HotMasterMix (5Prime) for amplification. But on EMP's website, the 16S protocol suggests Invitrogen's Platinum Hot Start PCR Master Mix (2x).

The Platinum SuperMix and Hot Start PCR Master Mix have a 6x and 1x fidelity in comparison to taq, respectively. Additionally, the former has exonuclease activity.

Is this discrepancy simply bc of the cost of the PCR kits (SuperMix is 2 times more expensive per reaction) and that EMP is collaboration so folks have different preferences?

Thanks! Taruna