Hello all,

I have been trying to follow a 2-stage PCR protocol used to amplify barcodes of a large yeast library, as per Nyugen et al. (2022) - https://link.springer.com/protocol/10.1007/978-1-0716-2257-5_22#Sec7 (section 3.3)

I have been working on this for the past 1.5 months now and have tried exploring all the variables from bead ratios to buffers to primers to template concentration. Although the 2-stage PCR seems to work every time now, the resulting yield after 2 cleanup steps is terribly low and cannot be sent for bulk sequencing.

Me and my advisor - both suspect cleanup steps as there wasn't anything wrong with the PCR itself. There are 2 bead cleanup steps after every stage of PCR:

1st cleanup - So the primers don't carry over to the 2nd stage of PCR.

2nd cleanup - remove excess primers and other contaminants.

The image attached would give some context to what I mean by low yield.