Matt:LabNotes/2016-9-14: Difference between revisions

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>Mzcai
(Created page with "==CA12k_Nov2014 V6 (RevComp of V4) Probe Production== * Expansion PCR and first time Production PCR ===Production PCR=== *V6 master mix {| {{tab...")
 
>Mzcai
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'''Program''' 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 22 -> 72C 2min -> 15C hold
'''Program''' 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold
*Stopped after 15 cycles
*Stopped after 15 cycles
[[File:012714_CA12kNov14_ProductionPCR_V6V8.JPG | 800px]]
[[File: | 800px]]


===EtOH Precipitation===
===EtOH Precipitation===
*6 5-ml tubes (with 8 wells of PCR product each) for V6 and 6 tubes for V8
*12 5-ml tubes (with 8 wells of PCR product each)
**800ul PCR product
**800ul PCR product
**2000ul 100% EtOH
**2000ul 100% EtOH
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*Eluted each column with 50ul and combined all V6 probes into one 1.5ml and all V8 probes into another
*Eluted each column with 50ul and combined all V6 probes into one 1.5ml and all V8 probes into another
*Measured concentration with Nanodrop:
*Measured concentration with Nanodrop:
 
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~290ul of V6 probes: 158.9 ng/ul => ~ 46 ug
~600ul of V6 probes: 158.9 ng/ul => ~ 46 ug


===Lambda Exonuclease Digestion===
===Lambda Exonuclease Digestion===

Revision as of 17:50, 15 September 2016

CA12k_Nov2014 V6 (RevComp of V4) Probe Production

Production PCR

  • V6 master mix
Components Volume (1X) Volume (50X)
First round amplicon CA12k_Nov14_V6 (10nM) 0.2 10
2X KAPA SYBG MM 50 2500
AP1V6U (100uM) 0.4 20
AP2V6 (100uM) 0.4 20
H2O 49 2450
Total 100 5000

Program 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold

  • Stopped after 15 cycles

[[File: | 800px]]

EtOH Precipitation

  • 12 5-ml tubes (with 8 wells of PCR product each)
    • 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 12 1.5mL tubes
  • Centrifuged 1.5ml tubes at 14,000rpm for 5 min at 4C
  • Discarded supernatant and air-dried for 5 min in hood
  • Resuspended DNA with 100ul H2O

Qia Column Purification

  • Purified 12 tubes of 100ul in 12 columns following Qiagen protocol
  • Eluted each column with 50ul and combined all V6 probes into one 1.5ml and all V8 probes into another
  • Measured concentration with Nanodrop: