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

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