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 | *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 | *Transferred DNA pellet (with 1ml pipette tip) to 8 1.5mL tubes | ||
*Centrifuged 1.5ml tubes at 14,000rpm for | *Centrifuged 1.5ml tubes at 14,000rpm for 15 min at 4C | ||
*Discarded supernatant and air-dried for | *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 | *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: | ||
~ | ~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 of Padlock Probes (V4) Matt:LabNotes/2014-11-19#Assembly_of_oligo_pool_for_ordering
Production PCR
- 10nM 1st round amplicons
- V4 master mix made in 2 5ml tubes
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