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 m (→Production PCR) |
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| Total||100||5000 | | Total||100||5000 | ||
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'''Program''' 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x | '''Program''' 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold | ||
*Stopped after 15 cycles | *Stopped after 15 cycles | ||
[[File: | [[File: | 800px]] | ||
===EtOH Precipitation=== | ===EtOH Precipitation=== | ||
* | *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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~ | ~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: