Hello I am also during SELEX right now. I also had some problems during the PCR part and I ended up asking Illumina for technical support. They pointed me to this protocol which you should consider reading: https://star-protocols.cell.com/protocols/1750

Since in R1 of SELEX the library is fully randomized, very little of it will be bound especially if your protein of interest is specific. You will see in that protocol I linked, to try and compensate for this most people start with more DNA library material for R1 (in the paper they do 1500 ng) then less for subsequent rounds (200 ng) as the high affinity sequences begin to dominate.

I would take your 11.67 nM DNA then do qPCR on it up to 40 cycles. See where the inflection point of the curve is and do that many cycles. What you can then do is multiple PCR reactions at the same conditions then pooled them all together to get enough DNA for the next round. As I said, R1 is the hardest so if you can make it past there, it gets easier.

Also keep in mind PCR amplification is exponential as the quantity doubles after every round. Thus even if you were to have 0.1% recovery your input DNA pre-PCR, after 15 cycles you will probably have more than when you started.

Hello Oshin,

It is because of the number of selected aptamer.

In the aptamer library, you have 10^10 ssDNA molecules. After first round, you might be 100 or less than ssDNA molecules specific to your target (meaning aptamer candidates). Therefore, you have to use all ssDNA obtained from first round to amplify by PCR. If you like, you can check our paper: https://www.researchgate.net/publication/283534928_Selection_of_Nucleic_Acid_Aptamers_Specific_for_Mycobacterium_tuberculosis

Good luck.

Best,

Erkan