Used a subset (10 samples) from the Ostrea lurida gDNA isolated 20150916 to prepare RAD libraries. This will be done to assess whether or not these samples, which appear to be heavily degraded, are viable for RAD-seq.
Followed the 2bRAD protocol (PDF) developed by Eli Meyer’s lab.
Prepared 1.2μg of each of the following samples in a volume of 10μL:
Google Sheet: 20150930_RADseq_DNA_calcs
Prepared a 150μM working stock of the SAM buffer needed for the restriction digestion by diluting 30μL of the supplied stock (500μM) in 70μL NanoPure H2O (total volume = 100μL). This working stock was stored @ -20C in FTR 209 in the “RAD-seq Reagents” box.
Prepared master mix for restriction enzyme reaction:
| REAGENT | SINGLE REACTION (μL) | x11 |
| DNA | 8 | NA |
| 10x Buffer R | 1.2μL | 13.2μL |
| 150μM SAM | 0.8μL | 8.8μL |
| AlfI | 0.5μL | 5.5μL |
| H2O | 1.5μL | 16.5μL |
Combined 4μL of the master mix with 8μL of each sample in 0.5mL snap cap tubes. Incubated @ 37C O/N in thermal cycler (no heated lid).