Geoduck Juvenile DNA Extractions (part4)
Extracted DNA from Juvenile Geoduck Tissue frozen at -80°C
Whole issue was previously homogenized using the Sample Homogenization Protocol
Sample list 20161025
- EPI-290
- EPI-289
- EPI-276
- EPI-275
- EPI-265
- EPI-264
- EPI-251
- EPI-250
- EPI-247
- EPI-246
- EPI-154
- EPI-153
- EPI-56
- EPI-55
- EPI-54
- EPI-53
- EPI-52
- EPI-51
- EPI-50
- EPI-49
- EPI-44
- EPI-43
- EPI-42
- EPI-41
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
- Saved samples at -80°C in 2 aliquots (6µl for gel and Qubit,~140µl)
DNA Quantification 20161026
- Used 1µl of sample and 199µl of Qubit Mix
- Ran Qubit dsDNA BR DNA Quantification Protocol
DNA Concentrations
| Sample.ID | Qubit Conc(ng/µl) | Dilution | Initial Conc(ng/µl) |
|---|---|---|---|
| EPI-290 | 7.4 | 1 | 7.4 |
| EPI-289 | 13.4 | 1 | 13.4 |
| EPI-276 | 21 | 1 | 21 |
| EPI-275 | 15 | 1 | 15 |
| EPI-265 | 17.5 | 1 | 17.5 |
| EPI-264 | 8.42 | 1 | 8.42 |
| EPI-251 | 16 | 1 | 16 |
| EPI-250 | 28.2 | 1 | 28.2 |
| EPI-247 | 5.98 | 1 | 5.98 |
| EPI-246 | 19.4 | 1 | 19.4 |
| EPI-154 | 19.5 | 1 | 19.5 |
| EPI-153 | 22.4 | 1 | 22.4 |
| EPI-56 | 21.4 | 1 | 21.4 |
| EPI-55 | 12.4 | 1 | 12.4 |
| EPI-54 | 15 | 1 | 15 |
| EPI-53 | 27.4 | 1 | 27.4 |
| EPI-52 | 9.82 | 1 | 9.82 |
| EPI-51 | 14.6 | 1 | 14.6 |
| EPI-50 | 5.24 | 1 | 5.24 |
| EPI-49 | 63.4 | 1 | 63.4 |
| EPI-44 | 14.8 | 1 | 14.8 |
| EPI-43 | 17.2 | 1 | 17.2 |
| EPI-42 | 3.18 | 1 | 3.18 |
| EPI-41 | 13.6 | 1 | 13.6 |
- a 140µl aliquot with an average of ~17ng/µl has ~2.4µg of DNA
DNA Quality 20161026
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-290
- EPI-289
- EPI-276
- EPI-275
- EPI-265
- EPI-264
- EPI-251
- EPI-250
- EPI-247
- EPI-246
- EPI-154
- EPI-153
- EPI-56
- EPI-55
- O Gene Ruler Mix SM 1173 Thermo Fisher 0.1µg/µl Range 100–10,000 bp
Gel Bottom
- O Gene Ruler Mix SM 1173 Thermo Fisher 0.1µg/µl Range 100–10,000 bp
- EPI-54
- EPI-53
- EPI-52
- EPI-51
- EPI-50
- EPI-49
- EPI-44
- EPI-43
- EPI-42
- EPI-41
- O Gene Ruler Mix SM 1173 Thermo Fisher 0.1µg/µl Range 100–10,000 bp
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 16 upper wells and 16 lower wells
- Once gel was set, Added 5µl of 6x loading dye to each sample of 5µl 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
- There is a clear difference in quality of samples between initial -80 or RNAlater storage
-
All samples have high molecular weight DNA present, -80 stored samples have less degradation
- Will continue to move forward with DNeasy DNA Extraction Protocol
Written on October 26, 2016