Shelly’s Notebook: Wed. Jul. 10, 2019 Salmon-Sea lice Zymo Pico Methyl prep + RRBS digest repeat

Zymo Pico Methyl kit prep

  • set up programs on PCR machine in 209 (called Zym1-4 under STRIGG folder)
  • Into a 48-well plate, I aliquoted out 20-50ng DNA and up to 20uL of nanopure H2O according to this sheet
  • I added 50ng of sea lice DNA:
    • Sea lice Female 1 = 67.2ng/uL (qubit HS, 7/10)
      • added 0.75uL + 19.25uL nanopure H2O
    • Sea lice Female 2 = 17ng/ul (qubit HS, 7/10)
      • added 3uL + 17uL nanopure H2O

Following Zymo pico methyl kit protocol section 1:

  • Added 130uL of lightening conversion reagent to all wells and incubated at 98C 8 min, 54C 1 hour, hold @4C
  • I combined all samples + 600uL of ***DNA binding buffer into Zymo DNA concentrator columns, spun, discarded supernatent
  • added 200uL desulphonation buffer to all columns and incubated at RT 20 min.

*** I realized during the incubation that instead of DNA binding buffer, this was suppose to be M-binding buffer!!! So I called Zymo tech support and was advised to not proceed with the kit because the yield could be poor in quantity and size and Zymo couldn’t guarentee the libraries would be representative. DNA binding buffer is not optimized for single stranded DNA (which after the conversion reagent, the DNA is). The DNA binding buffer can be used at a 6-7:1 ratio with ssDNA but that I not what I did. If I had kept the flow through I could have attempted to re-bind it to the column with the m-binding buffer.

SO, I took Zymo’s advice, and ordered more lightening conversion reagent to attempt the preps tomorrow. But I needed to repeat the digests to have enough DNA to start the preps with. BUMMER! But better that than get crappy data.

Repeating RRBS

  • Followed digest and size selection plan exactly as outlined here with the following exceptions:
    • I prepared reactions in a 48-well PCR plate
      • I first prepared the MSPI master mix and added it to all wells on ice. Then added DNA and water if needed
    • I created a program on the PCR machine in 209 for the incubations (called “RRBS” under the folder STRIGG).
    • MSPI digest incubated @37C for 1 hour
    • I paused the program to add 10uL of TAQ-a1 master mix/well
    • Then resumed the program to incubate at 65C for 30 min.
  • I measured DNA concentrations (S2 read 9.98ng/uL at the beginning and then 9.88 at the end)
    • DNA concentrations are here
  • I aliquoted 22-50ng salmon DNA into a 48-well plate following the volumes listed here and froze the plate at -20C

####plan for tomorrow

  • aliquot sea lice DNA into the plate
  • attempt all of Zymo pico methyl kit prep

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