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.