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4 Final Redo Sea Star Library Preps

4 Redo Sea Star Library Preps and Combine and Concentration

Using 1/4th reactions of the KAPA HyperPrep DNA Library Prep Kit

Samples are already sonicated

End Repair and A-tailing

  • Prepared end repair and a-tailing master mix on ice:
    • ERAT buffer 1.75μl * 4.5 = 7.875μl
    • ERAT enzyme 0.75μl * 4.5 = 3.375μl
  • Made 4 PCR strip tubes each with 12.5μl of 100ng sheared DNA
  • Added 2.5μl of ERAT master mix to each sample
  • Vortexed and spun down
  • Placed samples in thermocyler A-tailing program in JONP login (~ 1 hour)

Adapter Ligation

  • Prepared ligation master mix on ice (no vortexing of the ligase):
    • ligation buffer 7.5μl * 4.5 = 33.75μl
    • DNA ligase 2.5μl * 4.5 = 11.25μl
    • nuclease free water 1.25μl * 4.5 = 5.625μl
  • Added 11.25ul ligation master mix to each sample.
  • Added 1.25ul of planned adapter to each sample (see below). Adapters were added last to minimize adapter-adapter ligation
Library Number Adapter Number
14 2
34 10
37 1
45 9
  • Pipetted all samples up and down with the multichannel and spun down
  • Incubated on the shaker at room temp for 1-2 hours at 200rpm

0.8X Cleanup

  • Made fresh 80% EtOH
  • Took KAPA Pure Beads out of fridge beforehand to warm to room temp
  • After incubation at RT, added 22μl of KAPA pure beads to each sample and pipetted up and down at least 10 times to mix beads careful to avoid bubbles
  • Placed tubes on shaker at room temp for 15 minutes
  • Placed tubes on magnet plate and removed supernatant from tubes when it was fully clear not disturbing the beads
  • Added 200μl of 80% EtOH to each tube while still the magnet not disturbing the beads
  • Removed supernatant from each tube on the magnet plate without disturbing the beads
  • Added 200μl of 80% EtOH to each tube while still the magnet not disturbing the beads
  • Removed ALL the supernatant from each tube on the magnet plate without disturbing the beads. Extra EtOH blobs were removed with p20 pipette tips
  • Resuspended beads in 6μl 10mM Tris HCl pH 8 and incubated tubes on shaker for 5 minutes
  • Placed tubes back onto the magnet stand and removed 5ul supernatant when clear to new labeled PCR strip tubes

Library Amplification

  • Added 6.25ul KAPA HiFi to each sample tube
  • Added 0.75ul 503 and 0.75ul 703 to 14
  • Added 0.75ul 504 and 0.75ul 704 to 34
  • Added 0.75ul 505 and 0.75ul 705 to 37
  • Added 0.75ul 505 and 0.75ul 705 to 45
  • Vortexed and spun down samples
  • Placed in the thermocyler Genomic PCR 6 program

2 1X Cleanups

  • After PCR, added 12.5μl of KAPA pure beads to each sample and pipetted up and down at least 10 times to mix beads careful to avoid bubbles
  • Placed tubes on shaker at room temp for 15 minutes
  • Placed tubes on magnet plate and removed supernatant from tubes when it was fully clear not disturbing the beads
  • Added 200μl of 80% EtOH to each tube while still the magnet not disturbing the beads
  • Removed supernatant from each tube on the magnet plate without disturbing the beads
  • Added 200μl of 80% EtOH to each tube while still the magnet not disturbing the beads
  • Removed ALL the supernatant from each tube on the magnet plate without disturbing the beads. Extra EtOH blobs were removed with p20 pipette tips
  • Resuspended beads in 13μl 10mM Tris HCl pH 8 and incubated tubes on shaker for 5 minutes
  • Placed tubes back onto the magnet stand and removed 12.5ul supernatant when clear to new labeled PCR strip tubes
  • Added 12.5μl of KAPA pure beads to each sample and pipetted up and down at least 10 times to mix beads careful to avoid bubbles
  • Placed tubes on shaker at room temp for 15 minutes
  • Placed tubes on magnet plate and removed supernatant from tubes when it was fully clear not disturbing the beads
  • Added 200μl of 80% EtOH to each tube while still the magnet not disturbing the beads
  • Removed supernatant from each tube on the magnet plate without disturbing the beads
  • Added 200μl of 80% EtOH to each tube while still the magnet not disturbing the beads
  • Removed ALL the supernatant from each tube on the magnet plate without disturbing the beads. Extra EtOH blobs were removed with p20 pipette tips
  • Resuspended beads in 16μl 10mM Tris HCl pH 8 and incubated tubes on shaker for 5 minutes
  • Placed tubes back onto the magnet stand and removed 15ul supernatant when clear to new labeled PCR strip tubes

QC

Library Number Library Concentration
14 2.58
34 23.8
37 20.5
45 13

These weren’t great quantifications. I decided to combine the old and new libraries, concentrated them down to 16ul again, and qubit again.

  • Combined all 3 attempts of library 14, and the 2 attempts of 34, 37, and 45
  • Added either 40ul (14) or 30ul of KAPA pure beads to each tube (roughly 1X for all tubes) and pipette mixed
  • Placed tubes on shaker at room temp for 15 minutes
  • Placed tubes on magnet plate and removed supernatant from tubes when it was fully clear not disturbing the beads
  • Added 200μl of 80% EtOH to each tube while still the magnet not disturbing the beads
  • Removed supernatant from each tube on the magnet plate without disturbing the beads
  • Added 200μl of 80% EtOH to each tube while still the magnet not disturbing the beads
  • Removed ALL the supernatant from each tube on the magnet plate without disturbing the beads. Extra EtOH blobs were removed with p20 pipette tips
  • Resuspended beads in 16μl 10mM Tris HCl pH 8 and incubated tubes on shaker for 5 minutes
  • Placed tubes back onto the magnet stand and removed 15ul supernatant when clear to new labeled PCR strip tubes
  • Followed Qubit protocol for BR DNA
Library Number Library Concentration
14 5.3
34 27.8
37 29
45 18.7
  • I decided these were good enough
Written on August 3, 2021