Matt:LabNotes/2014-5-22: Difference between revisions

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***Why is it so low? Expected to be about 40%...
***Why is it so low? Expected to be about 40%...


*Noi suggests I may have had too much DNA in each tube for lambda digestion. She says 10ug DNA for 100ul reaction (15ug/150ul reaction)
<!-- To continue see: [[Matt:LabNotes/2013-5-13]] -->
<!-- To continue see: [[Matt:LabNotes/2013-5-13]] -->

Revision as of 18:52, 11 June 2014

Agi26k0gap Probe Production

  • Use 20nM aliquot of Agilent oligos in tube labeled "FISHYB V1 probes 5/07/13"

Probe Expansion PCR

  • Expansion PCR in 200ul total: 2 tubes of 100ul for 0bp gap probes with AP1V6 primers
' 0 gap probes (ul)
Agi26k (20nM) 10
2x Kapa SYBG qPCR MM 100
100uM pAP1V6U 0.8
100uM AP2V6 0.8
H2O 88.4
Total 200
  • Mixed thoroughly and then split into two tubes

94C 2min -> (94C 30sec -> 54C 45sec -> 72C 45sec) x 12 -> 72C 3min -> 15C hold

File:20140522 Agi26k0gapExpansionPCR.JPG

  • Purified with 2 Qiagen columns
  • Eluted with 40ul each tube and combined into two tubes (80ul total)
  • Nanodrop: 32.2 ng/ul -> 32.2ng/ul / (228bp*660Da/bp) = 214nM
  • Dilute 2.34ul in 47.66ul H2O
  • Final volumes:
    • 75ul of 214nM
    • 50ul of 10nM

Production PCR

  • 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:052314 Agi26k0gapProductionPCR.JPG

EtOH Precipitation

  • 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 overnight
  • 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

  • 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: 295.8 ng/ul => 65.1ug

Lambda Exonuclease Digestion

  • Divide each tube of 220ul into two 1.5ml tubes of 110ul with total amplicon of ~30ug
Components Volume (ul)
Amplicon 110
10X Lambda Exo Buffer 15
Lambda Exonuclease 10
H2O 15
total 150
  • Incubated at 37C for 1hr
  • Purified each of the tubes with 3 columns (6 columns total) of Zymo ssDNA/RNA kit
  • Eluted with 27ul each
  • Recombined and measured ssDNA with Nanodrop:
    • 49.2ng/ul x 160ul = 7.87ug (12% yield)
      • Why is it so low? Expected to be about 40%...
  • Noi suggests I may have had too much DNA in each tube for lambda digestion. She says 10ug DNA for 100ul reaction (15ug/150ul reaction)