Oligos from Peterson et al. (2011)

[maxbangs]

My lab is just starting up with the Peterson et al. (2011) protocol. I am testing restriction enzymes now and about to run the samples on a TapeStation.

The next step is to order the oligos for the adaptors/primers. Is there any thing I need to know about ordering the adaptors/primers or can I just order standard desalted oligos from IDT just like I do for Sanger sequencing?

[nucacidhunter]

If you are following their protocol you just need to order the oligos and anneal top and bottom oligos for various barcoded adapters and also common adapter. Bottom (barcoded) or top (common) oligos for adapters need to be 5' phosphorylated to allow for effective annealing to restricted DNA. 5' phosphorylation of oligos can be avoided if you incubate your PCR reaction for 3 min at 72°C (HotStart polymerase cannot be used) before cycling, but it may come in expense of even coverage and overlap of homologous loci among samples.

I am wondering if you could post TapeStation or gel photo of your single and double digests for some samples in this thread when you have them.

[maxbangs]

TapeStation results

So, I ran the samples on the TapeStation and am trying to figure out the number of loci per size selection. (I can post my results later once I figure out my size selection).

If I wanted to figure out the number of fragments for ddRAD in which the organisms genome size is ~2.5Gbps and the concentration of the whole sample is 19.6ng/ul and the size selection of 300+/- 36 bps yields 0.47ng/ul, then does the following math work at.

2,500,000,000bps * .47ng / 19.6ng /300bps / 2 = 99,915 loci

Thus if I do a size selection on the Pippin Prep of 376+/- 36bps (added 76bps for adapters) then I should get ~100,000loci. Is this right? If so then why is this more then double what I expected based on Peterson's estimates and in silico size selection? This is EcoRI by MspI by the way, I also have results for for other combinations across a few different species.