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.