ddRADseq success

Finally, success with the ddRADseq protocol!

Gel image of PCR titration of pooled DNA fragments for ddRADseq. Top row contains samples from NlaIII - MluCI double digestion. Bottom row contains samples from EcoRI - SPhI double digestion. Wells are (1) 100 bp ladder (2) 12 cycles (3) 14 cycles (4) 100 bp ladder (5) 16 cycles (6) 20 cycles (7) 100 bp ladder.

Image clearly shows that for the NlaIII - MluCI digestion and ligation, increased amplification with more cycles. This demonstrates that ligation of the P1 and P2 adapters occurred correctly.

For this de-bugging round, I started with a new P2 primer (ordered from IDT) that was not biotinylated. I’m not sure if this explains success as I suspect it may simply have to do with better lab technique, especially during the purification steps, so more DNA is retained at each step.