Geoduck Juvenile DNA Extractions

Extracted DNA from Juvenile Geoduck Tissue previously homogenized using the Sample Homogenization Protocol

Sample list

  • EPI-167
  • EPI-168
  • EPI-175
  • EPI-176
  • EPI-187
  • EPI-188
  • EPI-205
  • EPI-206
  • EPI-220
  • EPI-221
  • EPI-229
  • EPI-230

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 200µl 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 (6µl,~190µl)

DNA Concentrations

Sample.ID Qubit Conc(ng/µl) Dilution Initial Conc(ng/µl)
EPI-230 10.1 1 10.1
EPI-229 17.4 1 17.4
EPI-221 30.0 1 30.0
EPI-220 48.8 1 48.8
EPI-206 18.1 1 18.1
EPI-205 11.9 1 11.9
EPI-188 14.5 1 14.5
EPI-187 17.1 1 17.1
EPI-176 7.8 1 7.8
EPI-175 19.6 1 19.6
EPI-168 6.2 1 6.2
EPI-167 13.2 1 13.2
  • a 190µl aliquot with an average of ~17.5ng/µl has ~3µg of DNA
  • need to run gel quality check of DNA

DNA Quality 20161003

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 1

  • O Gene Ruler Mix SM 1173 Thermo Fisher 0.1µg/µl Range 100–10,000 bp
  • Test 1
  • Test 2
  • EPI-167
  • EPI-168
  • EPI-175
  • EPI-176
  • EPI-187
  • EPI-188
  • EPI-205
  • EPI-206
  • O Gene Ruler Mix SM 1173 Thermo Fisher 0.1µg/µl Range 100–10,000 bp

###Gel 2

  • O Gene Ruler Mix SM 1173 Thermo Fisher 0.1µg/µl Range 100–10,000 bp
  • EPI-220
  • EPI-221
  • EPI-229
  • EPI-230
  • O Gene Ruler Mix SM 1173 Thermo Fisher 0.1µg/µl Range 100–10,000 bp

Gel Setup

  • Added 0.75g of Agarose to 75ml of TAE and heated until clear
  • Added 6µl of Ethidium Bromide to gel
  • Poured gel with 12 upper wells and 8 lower wells
  • Once gel was set, Added 6µl of 6x loading dye to each sample of 6µl 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 45 minutes

Gel 1

Gel 1

Gel 2

Gel 2

Conclusions

  • Samples are more difficult to see in Gel 1
  • Appears to be large band >10,000 bp and some smear below
  • Samples in Gel 2 are more clear and appear to have relatively discrete bands with minimial smear
  • The data from Gel 2 suggest a good quality DNA extraction with minimial DNA degradation or contamination

  • Will move forward with DNeasy DNA Extraction Protocol
Written on September 30, 2016