Restriction Digest – Oly gDNA for RAD-seq w/AlfI

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:

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).

One comment

Leave a Reply

Your email address will not be published. Required fields are marked *

e.g. 0000-0002-7299-680X

You may use these HTML tags and attributes: <a href="" title=""> <abbr title=""> <acronym title=""> <b> <blockquote cite=""> <cite> <code> <del datetime=""> <em> <i> <q cite=""> <strike> <strong>