Excuse the bad pun… finally finished my proposal for the NSF Doctoral Dissertation Improvement Grant! Definitely one of the most time-consuming and stressful things I’ve done, as there are so many little parts that go into it and just one formatting error can get you disqualified without review. Check it out on my github here if you want an idea of what I have planned for the rest of my dissertation (pending funding, of course).

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.

Wednesday 9/30/15

Set up PCR of 10 samples for MiSeq test library.
  • Made 30 uL of 10mM Lib1 and Lib 2
    • 3+ 27 = 10 uM
  • Made 1 uM BC 2-10
    • 1 uL stock + 99 uL NFW
Master Mix
23 uL
10 mM (each) dNTPS
10 uM ILL-Lib1
10 uM ILL-Lib2
5X Q5 buffer
Q5 Taq polymerase
5 uL of 1 uM HT1 and appropriate BC to each
  1. SS2_3: BC1
  2. HC1_4: BC2
  3. HC1_3: BC3
  4. SS2_4: BC4
  5. SS2_5: BC5
  6. HC1_1 : BC6
  7. HC1_2: BC7
  8. NF1_1 : BC8
  9. NF1_2 : BC9
  10. NF1_3: BC10
 Made 5x recipe of TBE and diluted to 1x for a 2% medium sized gel. To make gel comb large enough for PCR product, I taped 2 combs together. Did not take into account that I still needed to run out a ladder, so only ran out 8 of the 10 samples.
Added 10 uL loading dye to 100 uL PCR products and was able to load 90 uL into wells. Ran out PCR at 110 V for 1 hour.
Gel of 2b-rad libraries 9-30-15. Pink arrow is product to be cut.
Gel of 2b-rad libraries 9-30-15. Pink arrow is product to be cut.
EtBr migrated to top of gel, so after cutting out the top bands (pink arrow), I soaked the bottom half of the gel in water with EtBR for 10 minutes before cutting out bottom bands.
Based on recommendation in the Matz 2b-rad protocol, I cut up gel slice into 4 pieces and put them in separate 1.5 mL tubes with 40 uL of NF water. These were put in 4degC fridge overnight. I’ve never done this type of gel extraction before so was a little skeptical.