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Australia Mytilus EecSeq Probe Synthesis Day 3

Second day of Stranded mRNA Seq Library Prep

All steps in are done side by side with Amy Zyck, continuing with sample tubes AL, FR, and MN, and Amy with TZ and RI

Following half reactions from the KAPA Biosystems Stranded mRNA-Seq Kit, also see the EeqSeq protocol

A-tailing and Ligation

  1. Made A-tailing after safe stopping point master mix:
    5.25μl nuclease-free water * 5.2 = 27.3μl
    0.75μl A-tailing buffer * 5.2 = 3.9μl
    1.5μl A-tailing enzyme * 5.2 = 7.8μl
  2. Took sample tubes out of the 4 degree and added 7.5μl of the A-tailing after safe stopping point master mix to each tube and pipetted to mix
  3. Put tubes in the thermocycler A-tailing program
  4. Made the Adapter Ligation Master Mix:
    8μl nuclease-free water * 5.2 = 41.6μl
    7μl ligation buffer * 5.2 = 36.4μl
    2.5μl DNA ligase * 5.2 = 13μl
  5. Added 17.5μl of the ligation master mix to each tube
  6. Added 2.5μl of the appropriate diluted working stock adapter to each tube:
    • AL - SAII
    • FR - NCO
    • MN - NCO
    • TZ - SAII
    • RI - NCO
  7. Pipetted to mix
  8. Put tubes in the thermomixer with the 96 well plate adapter so the tubes could be shaken at 300 speed and 20C for 1 hour of adapter ligation
  9. Added 35μl of room temperature PEG to each sample and pipetted to mix
  10. Placed on shaker for 15 minutes
  11. Made fresh 80% EtOH
  12. Placed tubes on magnet plate
  13. Removed 67μl of supernatant from each tube
  14. Added 100μl 80% EtOH to each tube
  15. Removed 100μl of supernatant from each tube
  16. Added 100μl 80% EtOH to each tube
  17. Removed ALL of the supernatant from each tube, using a p20 pipette tip to get rid of droplets
  18. Resuspended beads in 25μl of 10mM Tris HCl pH 8
  19. Placed tubes on shaker for 5 minutes
  20. Added 25μl of room temperature PEG to each tube and pipetted to mix
  21. Placed on shaker for 15 minutes
  22. Made fresh 80% EtOH
  23. Placed tubes on magnet plate
  24. Removed 45μl of supernatant from each tube
  25. Added 100μl 80% EtOH to each tube
  26. Removed 100μl of supernatant from each tube
  27. Added 100μl 80% EtOH to each tube
  28. Removed ALL of the supernatant from each tube, using a p20 pipette tip to get rid of droplets
  29. Resuspended beads in 11μl of 10mM Tris HCl pH 8
  30. Placed tubes on shaker for 5 minutes
  31. Placed tubes on magnet stand and removed 10μl of clear supernatant to new tubes
  32. Placed tubes in 4 degree overnight
Written on June 12, 2019