Tag Archives: Fidalgo Bay

DNA Isolation – Oly gDNA for BS-seq

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:

2NF1
2NF2
2NF3
2NF4
2NF5
2NF6
2NF7
2NF8
1NF11
1NF12
1NF13
1NF14
1NF15
1NF16
1NF17
1NF18

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

SAMPLE CONCENTRATION (ng/μL)
2NF1 76.4
2NF2 175
2NF3 690
2NF4 11.7
2NF5 142
2NF6 244
2NF7 25
2NF8 456
1NF11 182
1NF12 432
1NF13 155
1NF14 21
1NF15 244
1NF16 112
1NF17 25.2
1NF18 278

 

Will run samples on gel tomorrow to evaluate gDNA integrity.

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