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24 Sea Star WGS Library Preps

DNA Library Prep on 24 Sea Stars With 2.5 Min Sonication with 500ng, 100ng Going into DNA Prep

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

Sonication

  • Prepared 500ng of each sample in 80ul of 10mM tris HCL
Sample Library number 500ng ul 10mM tris HCL to 80ul
CHSY 003 1 14.5 65.5
CHSY 006 2 12.3 67.7
CPOI 005 3 12.5 67.5
CHSY 008 4 15.2 64.8
CPAB 006 5 10.5 69.5
CPAB2 008 6 12.6 67.4
CPBO 004 7 64.1 15.9
CPOI 006 8 12.6 67.4
CHSY 009 9 37.0 43.0
CPOI 004 10 9.6 70.4
CPAB2 001 11 14.7 65.3
CPAB2 004 12 25.1 54.9
CPBP 001 13 27.0 53.0
CPBO 008 14 40.3 39.7
CPBP-006 15 16.8 63.2
CPAB2 005 16 23.1 56.9
CPDB 001 17 28.7 51.3
CPOI 002 18 11.9 68.1
CHSB 009 19 24.8 55.2
CHSB 003 20 22.0 58.0
CPAB2 002 21 16.8 63.2
CPAB-001 22 18.9 61.1
Tank Escapee 23 5.0 75.0
CPAB 004 24 14.3 65.7
  • Ran sonicator for 2.5 minutes at 25% amplitude, 15 seconds on 15 seconds off. Samples were spun down after 1.25 minutes (general sonicator protocol)
  • 1X bead clean after sonication, followed protocol
  • Samples were re-suspended in 62.5ul 10mM tris HCl, this is 5*12.5, which is the input volume to the kit. I was then able to take 12.5ul from each sonicated sample and that should be ~100ng

End Repair and A-tailing

  • Prepared end repair and a-tailing master mix on ice:
    • ERAT buffer 1.75μl * 25 = 43.75μl
    • ERAT enzyme 0.75μl * 25 = 18.75μl
  • Made 31 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 * 25 = 187.5μl
    • DNA ligase 2.5μl * 25 = 62.5μl
    • nuclease free water 1.25μl * 25 = 31.25μl
  • 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
Sample Library number Adapter Number
CHSY 003 1 1
CHSY 006 2 2
CPOI 005 3 3
CHSY 008 4 4
CPAB 006 5 5
CPAB2 008 6 6
CPBO 004 7 7
CPOI 006 8 8
CHSY 009 9 9
CPOI 004 10 10
CPAB2 001 11 11
CPAB2 004 12 12
CPBP 001 13 1
CPBO 008 14 2
CPBP-006 15 3
CPAB2 005 16 4
CPDB 001 17 5
CPOI 002 18 6
CHSB 009 19 7
CHSB 003 20 8
CPAB2 002 21 9
CPAB-001 22 10
Tank Escapee 23 11
CPAB 004 24 12
  • 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

  • Made 2 master mixes for the two sets of index pairs
  • MM 2, library numbers 1-12
    • 6.25 KAPA HiFi * 13 = 81.25ul
    • 0.75ul 502 20uM primer * 13 = 9.75ul
    • 0,75ul 702 20uM primer * 13 = 9.75ul
  • MM 3, library numbers 13-24
    • 6.25 KAPA HiFi * 13 = 81.25ul
    • 0.75ul 503 20uM primer * 13 = 9.75ul
    • 0,75ul 703 20uM primer * 13 = 9.75ul
  • Vortexed and spun down mixes and kept on ice
  • Added 7.5ul of the appropriate mix to the strip tubes with the 5ul sample
  • Vortexed and spun down samples
  • Placed in the thermocyler Genomic PCR 6 program

1X Cleanup

  • 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 17μl 10mM Tris HCl pH 8 and incubated tubes on shaker for 5 minutes
  • Placed tubes back onto the magnet stand and removed 16ul supernatant when clear to new labeled PCR strip tubes

QC

Sample Library number Qubit Reading (ng/ul)
CHSY 003 1 65.4
CHSY 006 2 61.6
CPOI 005 3 57.8
CHSY 008 4 35.8
CPAB 006 5 55.6
CPAB2 008 6 101
CPBO 004 7 31.8
CPOI 006 8 71.8
CHSY 009 9 67
CPOI 004 10 103
CPAB2 001 11 54.6
CPAB2 004 12 52
CPBP 001 13 113
CPBO 008 14 need to redo
CPBP-006 15 126
CPAB2 005 16 116
CPDB 001 17 110
CPOI 002 18 103
CHSB 009 19 69
CHSB 003 20 124
CPAB2 002 21 80.8
CPAB-001 22 118
Tank Escapee 23 119
CPAB 004 24 88.6
  • D1000 TapeStation, 6 samples, check 14 to see if anything is there as well. Even though some of the libraries have super high quants, they look fine in size
Written on July 26, 2021