Matt:LabNotes/2014-7-1

From ZhangLabWiki
Jump to navigation Jump to search

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:2014-07-2 Agi26k0gapProductionPCR.JPG

EtOH Precipitation[edit]

  • 6 5-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 1hr
  • Centrifuged at 3000rpm at 4C for 30 min
  • 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: 138.8 ng/ul => 33.3 ug

Lambda Exonuclease Digestion[edit]

  • Divide into four pcr tubes of 55ul with total amplicon of ~10ug
Components Volume
Amplicon 55
10X Lambda Exo Buffer 10
Lambda Exonuclease 10
H2O 25
Total 100
  • Incubated at 37C for 1hr
  • Purified 2 of the tubes with of Zymo ssDNA/RNA columns and 2 of the tubes with Qiagen PCR columns
  • Eluted with 50ul each
  • Recombined and measured ssDNA with Nanodrop:
    • Zymo: 80.3 ng/ul x 100ul = ~8ug (8ug/16.65ug = 48% yield)
    • Qiagen: 61.9 ng/ul x 100ul = ~6.2ug (6.2ug/16.65ug = 37% yield)
    • Zymo is more efficient and this time had no trouble with efficiency
  • Used new Lambda enzyme and buffer, maybe that is why it had higher yield