Used a subset (10 samples) from the Ostrea lurida gDNA isolated 20150917 to prepare RAD libraries.
Followed the 2bRAD protocol (PDF) developed by Eli Meyer’s lab.
Prepared 9.0μg of each of the following samples in a volume of 10μL:
Google Sheet: 20151009_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 2hrs. in thermal cycler (PTC-200; no heated lid). Heat inactivated the digest @ 65C for 10mins.