Geoduck Juvenile DNA Extractions (part3)
Extracted DNA from Juvenile Geoduck Tissue frozen at -80°C
Whole issue was previously homogenized using the Sample Homogenization Protocol
Sample list 20161007
- EPI-299
- EPI-298
- EPI-272
- EPI-271
- EPI-243
- EPI-242
- EPI-227
- EPI-226
- EPI-209
- EPI-208
- EPI-185
- EPI-184
- EPI-170
- EPI-169
- EPI-162
- EPI-161
- EPI-145
- EPI-143
- EPI-120
- EPI-119
- EPI-113
- EPI-111
- EPI-104
- EPI-103
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 20161010
- 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-299 | 17.6 | 1 | 17.6 |
| EPI-298 | 3.48 | 1 | 3.48 |
| EPI-272 | 5.58 | 1 | 5.58 |
| EPI-271 | 2.10 | 1 | 2.10 |
| EPI-243 | 3.76 | 1 | 3.76 |
| EPI-242 | 6.20 | 1 | 6.20 |
| EPI-227 | 31.6 | 1 | 31.6 |
| EPI-226 | 13.1 | 1 | 13.1 |
| EPI-209 | 22.0 | 1 | 22.0 |
| EPI-208 | 31.0 | 1 | 31.0 |
| EPI-185 | 18.6 | 1 | 18.6 |
| EPI-184 | 7.46 | 1 | 7.46 |
| EPI-170 | 11.2 | 1 | 11.2 |
| EPI-169 | 14.8 | 1 | 14.8 |
| EPI-162 | 12.5 | 1 | 12.5 |
| EPI-161 | 2.26 | 1 | 2.26 |
| EPI-145 | 9.64 | 1 | 9.64 |
| EPI-143 | 2.46 | 1 | 2.46 |
| EPI-120 | 16.5 | 1 | 16.5 |
| EPI-119 | 17.6 | 1 | 17.6 |
| EPI-113 | 18.8 | 1 | 18.8 |
| EPI-111 | 55.6 | 1 | 55.6 |
| EPI-104 | 9.28 | 1 | 9.28 |
| EPI-103 | 3.50 | 1 | 3.50 |
- a 140µl aliquot with an average of ~14ng/µl has ~1.9µg of DNA
DNA Quality 20161010
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-299
- EPI-298
- EPI-272
- EPI-271
- EPI-243
- EPI-242
- EPI-227
- EPI-226
- EPI-209
- EPI-208
- EPI-185
- EPI-184
- EPI-170
- EPI-169
Gel Bottom
- O Gene Ruler Mix SM 1173 Thermo Fisher 0.1µg/µl Range 100–10,000 bp
- EPI-162
- EPI-161
- EPI-145
- EPI-143
- EPI-120
- EPI-119
- EPI-113
- EPI-111
- EPI-104
- EPI-103
- EPI-128 (from 20161004)
- EPI-127 (from 20161004)
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 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
- Smear may be from residual degraded RNA
-
All samples have high molecular weight DNA present
- Will continue to move forward with DNeasy DNA Extraction Protocol
Written on October 7, 2016