Thursday 10/1/15

Took gel slices out of 4degC and they did not look dissolved. Pipetted out the supernatant from each and quantified with HS Qubit. Qubit said the DNA concentration was too low to be recorded, so I added the supernatant back to the respective tubes with gel and did a gel extraction with the Qiagen kit. Added 10 uL of 3M sodium acetate to each sample to bring the pH down. Eluted in 40 uL of provided elution buffer after letting buffer sit in column for 4 minutes. Quantified with HS Qubit:
  • SS2_3: 0.21
  • HC1_4: 0.283
  • HC1_3: .69
  • SS2_4: .281
  • SS2_5: .983
  • HC1_1 : .271
  • HC1_2: .45
  • NF1_1 : .372
In order to multiplex these at equal concentrations, I multiplied the lowest concentration by 38 (the volume after Qubit). I then divided this number by each of the other concentrations to get the volume to add to the pool. Calculations are shown on the 2nd sheet of the Common Garden Samples master list. To concentrate the pooled library, I used a single column from Qiagen Quiaquick PCR Kit. This column was eluted in 30 uL after 5 minute incubation, and was quantified via Qubit at 4.19 ng/uL.
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