Sam's Notebook

DNA Isolation - Oly gDNA for BS-seq

by ["kubu4"] 1 min read

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:

<td data-sheets-value="[null,2,"2NF1"]" >2NF1 </td> <td data-sheets-value="[null,2,"2NF2"]" >2NF2 </td> <td data-sheets-value="[null,2,"2NF3"]" >2NF3 </td> <td data-sheets-value="[null,2,"2NF4"]" >2NF4 </td> <td data-sheets-value="[null,2,"2NF5"]" >2NF5 </td> <td data-sheets-value="[null,2,"2NF6"]" >2NF6 </td> <td data-sheets-value="[null,2,"2NF7"]" >2NF7 </td> <td data-sheets-value="[null,2,"2NF8"]" >2NF8 </td> <td data-sheets-value="[null,2,"1NF11"]" >1NF11 </td> <td data-sheets-value="[null,2,"1NF12"]" >1NF12 </td> <td data-sheets-value="[null,2,"1NF13"]" >1NF13 </td> <td data-sheets-value="[null,2,"1NF14"]" >1NF14 </td> <td data-sheets-value="[null,2,"1NF15"]" >1NF15 </td> <td data-sheets-value="[null,2,"1NF16"]" >1NF16 </td> <td data-sheets-value="[null,2,"1NF17"]" >1NF17 </td> <td data-sheets-value="[null,2,"1NF18"]" >1NF18 </td>

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

<td data-sheets-value="[null,2,"2NF1"]" >**SAMPLE** </td> <td data-sheets-value="[null,2,"2NF1"]" >2NF1 </td> <td data-sheets-value="[null,2,"2NF2"]" >2NF2 </td> <td data-sheets-value="[null,2,"2NF3"]" >2NF3 </td> <td data-sheets-value="[null,2,"2NF4"]" >2NF4 </td> <td data-sheets-value="[null,2,"2NF5"]" >2NF5 </td> <td data-sheets-value="[null,2,"2NF6"]" >2NF6 </td> <td data-sheets-value="[null,2,"2NF7"]" >2NF7 </td> <td data-sheets-value="[null,2,"2NF8"]" >2NF8 </td> <td data-sheets-value="[null,2,"1NF11"]" >1NF11 </td> <td data-sheets-value="[null,2,"1NF12"]" >1NF12 </td> <td data-sheets-value="[null,2,"1NF13"]" >1NF13 </td> <td data-sheets-value="[null,2,"1NF14"]" >1NF14 </td> <td data-sheets-value="[null,2,"1NF15"]" >1NF15 </td> <td data-sheets-value="[null,2,"1NF16"]" >1NF16 </td> <td data-sheets-value="[null,2,"1NF17"]" >1NF17 </td> <td data-sheets-value="[null,2,"1NF18"]" >1NF18 </td>
**CONCENTRATION (ng/μL)**
76.4
175
690
11.7
142
244
25
456
182
432
155
21
244
112
25.2
278

Will run samples on gel tomorrow to evaluate gDNA integrity.