Matt:LabNotes/2014-7-17

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Agi26k0gap Probe Production[edit]

Production PCR[edit]

  • Make Master Mix for 46 well (6 strips)
  • 0 gap first round amplicon -> V6 primers
Components 1x rxn (ul) 50x rxn (ul)
First round amplicon (10nM) 0.2 10
2x Kapa SYBR MM 50 2500
100uM pAP1V6U 0.4 20
100uM AP2V6 0.4 20
H2O 49 2450
Total 100 5000

98C 30sec -> (98C 10sec -> 55C 20sec -> 72C 30sec) x 15 -> 72C 2min -> 15C hold

File:2014-07-17 Agi26k0gapProductionPCR.JPG

EtOH Precipitation[edit]

  • 6 15-ml tubes (with 8 wells of PCR product each) for 0gap
    • 800ul PCR product
    • 2000ul 100% EtOH
    • 2.7ul GlycoBlue
    • 80ul 3M NaOAc pH 5.2-5.5
  • Vortexed and put in -80C for overnight
  • Centrifuged at 3000rpm at 4C for 30min
  • Discarded supernatant and added 800ul of cold 80% EtOH
  • Transferred DNA pellet (with 1ml pipette tip) to 6 1.5mL tubes
  • Centrifuged 1.5ml tubes at 15,000rpm for 5 min on table-top centrifuge
  • Discarded supernatant and air-dried for 5 min in hood
  • Resuspended DNA with 100ul H2O

Qia Column Purification[edit]

  • Purified 6 tubes of 100ul in 6 columns following Qiagen protocol
  • Eluted each column with 40ul and combined all probes into one 1.5ml
  • Measured concentration with Nanodrop:
  • ~220ul of Agi26k0gap probes: 352.2 ng/ul => 77.5 ug
    • Every time I tried to measure sample it gave me a warning message that there might be a bubble or unclean pedastal, but consistently measured ~355ng/ul (all 3 times)
    • In between the 3 sample measurements I cleaned and measured a blank water sample and got +/-2ng/ul with no warning message
      • Next day re-measured and got 355.5 ng/ul with no error


Lambda Exonuclease Digestion[edit]

  • Divide into eight pcr tubes of 27.5ul with total amplicon of ~10ug each
    • Four with old Lambda and Four with new Lambda
      • Fourth tube of each set had extra 2.5ul amplicon
Components Volume
Amplicon 27.5
10X Lambda Exo Buffer 10
Lambda Exonuclease 10
H2O 52.5
Total 100
  • Incubated at 37C for 1hr
  • Purified with of Zymo ssDNA/RNA columns
  • Eluted with 40ul each
  • Combined the three tubes with 27.5ul amplicon to measure and compare
  • Recombined and measured ssDNA with Nanodrop:
    • New Lambda: 69.5ng/ul x 120ul = 8.34ug
    • Old Lambda: 65.3ng/ul x 120ul = 7.84ug
      • Estimate total yield: ~67ng/ul x 320ul = 21.44ug (21.44/38.75 = 55.3%)
  • Result: Negligible difference between old and new lambda
    • Not sure what was causing low yield during Lambda digestion Matt:LabNotes/2014-6-24
    • Checked Qiagen vs Zymo columns and that wasn't the reason either
      • Whatever it was, things seem to work well now