Geoduck Juvenile DNA Extractions (part2)
Extracted DNA from Juvenile Geoduck Tissue frozen at -80°C
Whole issue was previously homogenized using the Sample Homogenization Protocol
Sample list
- EPI-303
- EPI-302
- EPI-283
- EPI-282
- EPI-258
- EPI-257
- EPI-236
- EPI-235
- EPI-215
- EPI-214
- EPI-200
- EPI-199
- EPI-194
- EPI-193
- EPI-182
- EPI-181
- EPI-160
- EPI-159
- EPI-152
- EPI-151
- EPI-136
- EPI-135
- EPI-128
- EPI-127
DNA Extractions
- added ~20mg of tissue to 180µl of Buffer ATL and 20µl of Proteinase K
- Proceeded with DNA Extraction Protocol
- Did not do the RNase treatment
- Eluted in 150µl of AE Buffer
DNA Quantification
- Used 1µl of sample and 199µl of Qubit Mix
- Ran Qubit dsDNA BR DNA Quantification Protocol
- Saved samples at -80°C in 2 aliquots (3µl for gel,~140µl)
DNA Concentrations
| Sample.ID | Qubit Conc(ng/µl) | Dilution | Initial Conc(ng/µl) |
|---|---|---|---|
| EPI-303 | 32.4 | 1 | 32.4 |
| EPI-302 | 32.8 | 1 | 32.8 |
| EPI-283 | 5.50 | 1 | 5.50 |
| EPI-282 | 10.3 | 1 | 10.3 |
| EPI-258 | 3.26 | 1 | 3.26 |
| EPI-257 | 15.4 | 1 | 15.4 |
| EPI-236 | 8.66 | 1 | 8.66 |
| EPI-235 | 10.6 | 1 | 10.6 |
| EPI-215 | 22.6 | 1 | 22.6 |
| EPI-214 | 13.3 | 1 | 13.3 |
| EPI-200 | 21.2 | 1 | 21.2 |
| EPI-199 | 21.8 | 1 | 21.8 |
| EPI-194 | 11.7 | 1 | 11.7 |
| EPI-193 | 9.26 | 1 | 9.26 |
| EPI-182 | 20.8 | 1 | 20.8 |
| EPI-181 | 40.8 | 1 | 40.8 |
| EPI-160 | 10.8 | 1 | 10.8 |
| EPI-159 | 21.4 | 1 | 21.4 |
| EPI-152 | 16.8 | 1 | 16.8 |
| EPI-151 | 11.8 | 1 | 11.8 |
| EPI-136 | 18.2 | 1 | 18.2 |
| EPI-135 | 20.2 | 1 | 20.2 |
| EPI-128 | 29.8 | 1 | 29.8 |
| EPI-127 | 35.2 | 1 | 35.2 |
- a 140µl aliquot with an average of ~18.5ng/µl has ~2.8µg of DNA
- need to run gel quality check of DNA
## DNA Quality 20161006 Ran a quality check of DNA using a 1% TAE gel in 1x TAE running buffer
Gel Preparation
TAE
- 40 mM Tris (pH 7.6)
- 20 mM acetic acid
- 1 mM EDTA
Samples
Gel Top
- O Gene Ruler Mix SM 1173 Thermo Fisher 0.1µg/µl Range 100–10,000 bp
- EPI-303
- EPI-302
- EPI-283
- EPI-282
- EPI-258
- EPI-257
- EPI-236
- EPI-235
- EPI-215
- EPI-214
- EPI-200
Gel Bottom
- O Gene Ruler Mix SM 1173 Thermo Fisher 0.1µg/µl Range 100–10,000 bp
- EPI-199
- EPI-194
- EPI-193
- EPI-182
- EPI-181
- EPI-160
- EPI-159
- EPI-152
- EPI-151
- EPI-136
- EPI-135
- EPI-128
- EPI-127
Gel Setup
- Added 1.5g of Agarose to 150ml of TAE and heated until clear
- Added 12µl of Ethidium Bromide to gel
- Poured gel with 12 upper wells and 12 lower wells
- Once gel was set, Added 3µl of 6x loading dye to each sample of 3µl of DNA (6x purple loading dye #B70245 New Endland BioLabs)
- Added 5µl of the sample-loading dye mix to each well
- Ran gel at 100v for 60 minutes
Gel 1
Conclusions
- Samples are more difficult to see then on actual gel
- Appears to be large band >10,000 bp and some smear below
-
All samples have high molecular weight DNA present
- Will continue to move forward with DNeasy DNA Extraction Protocol
Written on October 4, 2016