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Second Extraction Pentagona HMW Size Selection with BluePippin

Using the BluePippin High Pass Plus Cassette, >15 kb DNA size collections to further refine the second HMW pentagona DNA extraction to above 15,000bp fragments only

Sample Prep

11/12/19

  • Took out electrophoresis buffer, marker, and loading solution from the 4 degree fridge at least 30 minutes before use to get to room temp
  • Prepared PCR tubes with all of each sample. This was:
    • 27.5µl PG3
    • 26µl PG4
  • Added 2.5µl and 4µl of TE buffer to each sample to bring up to 30µl each respectively
  • Added 10µl of room temp loading solution to each sample and pipetted to mix
  • Realized that I couldn’t do both at the same time, so PG4 with loading solution was placed in the 4 degree fridge until PG3 was finished in the Pippin

BluePippin Prep

11/12/19

  • Note two half used cassettes were used, meaning two runs each with one sample and one marker were done for these 2 samples
  • Brought p20, p200, cassette, tips, solutions, and samples up to the GSC
  • Edited the protocol for this cassette:
    • Selected “range” for lane 2
    • Selected reference for lane 1
    • Clicked “apply reference to all lanes”
    • Unclicked range and set reference to off for lanes 5, 4, and 3
    • Set start as 15,000bp and end as 150,000bp so the target was 82,500bp for lane 1
    • Saved protocol as MES_15kb_HighPass
  • Calibrated the optics feature
  • Unwrapped cassette, removed foil seals I have used to save it from last time, and looked for problems with the gel: none
  • Checked amount of buffer in reservoirs: all well full
  • Removed all buffer from elution modules (~80µl) and replaced it with fresh electrophoresis buffer 80µl. Do this in all elution modules even ones not using
  • Sealed elution modules with sicky provided with the cassettes
  • Closed lid and performed continuity test: failed only in lane 5 Previously I had talked to Sage Science technicians about reusing cassettes and they said that if wells/lanes that were used before fail the continuity test it’s fine to use the other lanes that pass and that you haven’t used before
  • Removed 40µl of buffer from 2 and 1 sample wells
  • Added 40µl marker U1 to well 2
  • Added 40µl PG3 to well 1
  • Closed lid and pressed start
  • Program ran until idle, 2.5 hours later
  • Set timer for 45 min to let all sample elute and waited to remove until after that
  • After 45 min, removed sticker and saved all the liquid in elution modules 1
  • Filled elution modules 1 with 80µl 0.1% Tween and waited 1 minute, then removed and saved that liquid as well
  • Put those samples in the 4 degree fridge
  • Followed the exact same steps above with the second half used cassette and with sample PG4

QC

Sample Standard 1 Standard 2 Reading 1 ng/µl Reading 2 ng/µl Average ng/µl
PG3 Top 47 24579 16.5 16.5 16.5
PG3 Bottom 47 24579 19.6 19.6 19.6
PG4 Top 47 24579 34.2 34.2 34.2
PG4 Bottom 47 24579 33.6 33.8 33.7
PG3 Tween Top 47 24579 1.56 1.58 1.57
PG3 Tween Bottom 47 24579 1.71 1.73 1.72
PG4 Tween Top 47 24579 0.47 0.478 0.474
PG4 Tween Bottom 47 24579 0.516 0.524 0.54
  • Ran D5000 tapestation on the elution module liquid (not the tween washes) 2 3

full report

0.45X Cleanup of Elution Liquid and Tween Washes

11/13/19 and 11/14/19

  • Made fresh 80% EtOH before starting
  • Took KAPA Pure Beads out of fridge about 30 minutes before they were needed
  • Used pipettes to determine how much volume of sample out of the BluePippin there was:
    • PG3: 73µl so .45X is 32.85µl of KAPA Pure Beads
    • PG4: 74µl so .45X is 33.3µl of KAPA Pure Beads
    • PG3T: 78µl so .45X is 35.1µl of KAPA Pure Beads
    • PG4T: 80µl so .45X is 36µl of KAPA Pure Beads
  • Added the above amount of beads to each sample very gently then flicked the tubes gently until a homogeneous distribution of beads was obtained and then tubes were spun down briefly in a minifuge
  • Placed tubes on shaker for 20 minutes at room temp at 300rpm
  • Then placed the tubes on the magnet rack and waited 10 minutes
  • Removed the clear supernatant and SAVED as the sample name and “supernatant”
  • Added 200µl of fresh 80% EtOH to each tube gently then waited 30 seconds
  • Removed and SAVED as the sample name and “wash 1”
  • Added 200µl of fresh 80% EtOH to each tube gently then waited 30 seconds
  • Removed and SAVED as the sample name and “wash 2”
  • Spun sample tubes briefly in tabletop minifuge and placed back on the magnet rack
  • Used p20 to get the last liquid out of the tube, just a few µl
  • Did not let samples rest really, removed tubes from rack and added 50µl of nuclease free water to each tube by gently dripping
  • Flicked tubes until the pellets resuspended and then a little further, looked at beads very closely and tried to flick away any small clumps, and then briefly spun down because liquid was all on the lids
  • Placed tubes closed on sample rack still at room temp for 20 minutes
  • After then placed on shaker at room temp for afternoon and overnight at 300prm
  • The next morning, spun down tubes briefly because some evaporation, also the bead distribution was different between the real samples and the washes: 3
  • Placed tubes on the magnet rack for 10 minutes
  • Removed the clear supernatant and placed into new labeled sample tubes
  • Added 50µl of nuclease-free water to the beads again and saved in 4 degree fridge

High Sensitivity Qubit of top and bottom of sample tubes and washes
Flipped tube end over end to try to mix sample before taking 1µl each to Qubit

Sample Standard 1 Standard 2 Reading 1 ng/µl Reading 2 ng/µl Average ng/µl
PG3 Top 41.58 24660 16.3 16.3 16.3
PG3 Bottom 41.58 24660 17.1 17.1 17.1
PG4 Top 41.58 24660 33.4 33.6 33.5
PG4 Bottom 41.58 24660 30.6 30.6 30.6
PG3 Tween Top 41.58 24660 0.69 0.656 0.67
PG3 Tween Bottom 41.58 24660 0.656 0.662 0.658
PG4 Tween Top 41.58 24660 0.182 0.19 0.186
PG4 Tween Bottom 41.58 24660 0.178 0.182 0.18
Written on November 12, 2019