Australia Mytilus EecSeq Probe Synthesis Day 7

Restriction Digest of Adapters, Mung bean Nuclease Treatment, and Biotinylation

Cleanup and visualization of probes were done the next day, but are included here for clarity

Continuing with Amy Zyck and Jacob Green

1. 1μg of DSN treated library is a good amount for the restriction digest reaction to remove the adapters. The quantities after the secondary 8 cycle PCR are good enough for this and no further PCR was needed
2. Determined μl needed of each sample needed for 1μg:
   • 7.63μl sample 1
   • 5.85μl sample 2
3. Determined volume of 10mM Tris HCl needed to add up to 12.25μl from volumes above:
   • 4.37μl for sample 1
   • 6.4μl for sample 2
4. Created 2 new PCR tubes with the appropriate amount of 10mM Tris HCl and post-PCR DSN treated libraries to be 1μg in 12.25μl
   note here that accidentally 4.37μl of Tris were added to sample 1 instead of a new tube, based on the previous qubit a new volume for 1μg was calculated to be: 9.30μl post-PCR DSN treated library and 2.95μl Tris
5. Made a master mix for the restriction digest:
   • 4μl 10X cutsmart buffer * 2.2 = 8.8μl
   • 1μl SAII Enzyme * 2.2 = 2.2μl
   • 1μl NCO Enzyme * 2.2 = 2.2μl
   • 21.75μl nuclease-free water * 2.2 = 47.85μl
6. Prepared reactions on ice beads, added 27.75μl of the RE master mix to the two new tubes with 12.25μl diluted post-PCR DSN treated libraries
7. Placed in thermocycler RE digest program: 37°C for 4 hours and 80°C for 20 minutes
8. Prepared mung bean master mix with ~10 minutes left on the digestion:
   • 4.5μl mung bean nuclease buffer * 2.2 = 9.9μl
   • .5μl mung bean enzyme * 2.2 = 1.1μl
9. Added 5μl of mung bean master mix to each tube out of the digestion, pipetted to mix, and incubated at 30C for 30 minutes in the thermocycler program MBN

Written on July 1, 2019