Matt:LabNotes/2015-5-11: Difference between revisions

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>Mzcai
(Created page with "==CA12k_Nov2014_V4 Probe Production== *Production of Padlock Probes (V4) Matt:LabNotes/2014-11-19#Assembly_of_oligo_pool_for_ordering ===Production PCR=== *[[Matt:LabNote...")
 
>Mzcai
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**120ul 3M NaOAc pH 5.2-5.5
**120ul 3M NaOAc pH 5.2-5.5


*Vortexed and put in -80C for overnight <!--
*Vortexed and put in -80C for overnight  
*Centrifuged at 3000rpm at 4C for 30 min
*Centrifuged at 13000rpm at 4C for 20 min (in basement ultracentrifuge)
*Discarded supernatant and added 800ul of cold 80% EtOH
*Discarded supernatant and added 800ul of cold 80% EtOH
*Transferred DNA pellet (with 1ml pipette tip) to 12 1.5mL tubes
*Transferred DNA pellet (with 1ml pipette tip) to 8 1.5mL tubes
*Centrifuged 1.5ml tubes at 14,000rpm for 5 min at 4C
*Centrifuged 1.5ml tubes at 14,000rpm for 15 min at 4C
*Discarded supernatant and air-dried for 30 min in hood
*Discarded supernatant and air-dried for 10 min in hood
*Resuspended DNA with 100ul H2O
*Resuspended DNA with 100ul H2O


===Qia Column Purification===
===Qia Column Purification===
*Purified 12 tubes of 100ul in 12 columns following Qiagen protocol
*Purified 8 tubes of 100ul in 8 columns following Qiagen protocol
*Eluted each column with 50ul and combined all into one 1.5ml
*Eluted each column with 50ul and combined all into one 1.5ml
*Measured concentration with Nanodrop:
*Measured concentration with Nanodrop:


~600ul of V4 probes: 59.8 ng/ul => ~35.9 ug
~400ul of V4 probes: ng/ul => ~ ug
 
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===Lambda Exonuclease Digestion===
===Lambda Exonuclease Digestion===
*Divide into 6 pcr tubes of ~100ul with total amplicon of <10ug each
*Divide into 6 pcr tubes of ~100ul with total amplicon of <10ug each

Revision as of 19:39, 12 May 2015

CA12k_Nov2014_V4 Probe Production

Production PCR

Components Volume (1X) Volume (100X)
First round amplicon CA12k_Nov14_V4 (10nM) 0.2 20
2X KAPA SYBG MM 50 5000
AP1V4U (100uM) 0.4 40
AP2V4 (100uM) 0.4 40
H2O 49 4900
Total 100 10000

Program 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold File:20150511 CA12kNov2014 V4 ProductionPCR.JPG

EtOH Precipitation

  • 8 5-ml tubes (with 12 wells of PCR product each) for V4
    • 1200ul PCR product
    • 3000ul 100% EtOH
    • 3ul GlycoBlue
    • 120ul 3M NaOAc pH 5.2-5.5
  • Vortexed and put in -80C for overnight
  • Centrifuged at 13000rpm at 4C for 20 min (in basement ultracentrifuge)
  • Discarded supernatant and added 800ul of cold 80% EtOH
  • Transferred DNA pellet (with 1ml pipette tip) to 8 1.5mL tubes
  • Centrifuged 1.5ml tubes at 14,000rpm for 15 min at 4C
  • Discarded supernatant and air-dried for 10 min in hood
  • Resuspended DNA with 100ul H2O

Qia Column Purification

  • Purified 8 tubes of 100ul in 8 columns following Qiagen protocol
  • Eluted each column with 50ul and combined all into one 1.5ml
  • Measured concentration with Nanodrop:

~400ul of V4 probes: ng/ul => ~ ug