Need DNA to prep our own libraries for bisulfite-treated high-throughput sequencing (BS-seq).

Isolated gDNA from the following tissue samples stored in RNAlater (tissue was not weighed) using DNAzol:

The sample coding breaks down as follows (see the project wiki for a full explanation):

2NF#

2 = Oysters outplanted in Fidalgo Bay

NF = Broodstock originated in Fidalgo Bay

# = Sample number

1NF#

1 = Oysters outplanted in Oyster Bay

NF = Broodstock originated in Fidalgo Bay

# = Sample number

DNA was isolated in the following manner:

• Homogenized tissues in 500μL of DNAzol (Molecular Research Center; MRC).
• Added additional 500μL of DNAzol.
• Added 10μL of RNase A (10mg/mL, ThermoFisher); incubated 10mins @ RT.
• Added 300μL of chloroform and mixed moderately fast by hand.
• Incubated 5mins @ RT.
• Centrifuged 12,000g, 10mins, RT.
• Transferred aqueous phase to clean tube.
• Added 500μL of 100% EtOH and mixed by inversion.
• Pelleted DNA 5,000g, 5mins @ RT.
• Performed 3 washes w/70% EtOH.
• Dried pellet 3mins.
• Resuspended in 100μL of Buffer EB (Qiagen).
• Centrifuged 12,000g, 10mins, RT to pellet insoluble material.
• Transferred supe to clean tube.

The samples were quantified using the Qubit dsDNA BR reagents (Invitrogen) according to the manufacturer’s protocol and used 1μL of sample for measurement.

Results:

Qubit data (Google Sheet): 20151216_Oly_gDNA_qubit_quants

Will run samples on gel tomorrow to evaluate gDNA integrity.