Matt:LabNotes/2014-7-17: Difference between revisions
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>Mzcai mNo edit summary |
>Mzcai mNo edit summary |
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===EtOH Precipitation=== | ===EtOH Precipitation=== | ||
*6 | *6 15-ml tubes (with 8 wells of PCR product each) for 0gap | ||
**800ul PCR product | **800ul PCR product | ||
**2000ul 100% EtOH | **2000ul 100% EtOH | ||
Line 36: | Line 36: | ||
**80ul 3M NaOAc pH 5.2-5.5 | **80ul 3M NaOAc pH 5.2-5.5 | ||
*Vortexed and put in -80C for | *Vortexed and put in -80C for overnight | ||
*Centrifuged at 3000rpm at 4C for | *Centrifuged at 3000rpm at 4C for 30min | ||
*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 6 1.5mL tubes | *Transferred DNA pellet (with 1ml pipette tip) to 6 1.5mL tubes | ||
Line 50: | Line 50: | ||
~220ul of Agi26k0gap probes: 138.8 ng/ul => 33.3 ug | ~220ul of Agi26k0gap probes: 138.8 ng/ul => 33.3 ug | ||
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===Lambda Exonuclease Digestion=== | ===Lambda Exonuclease Digestion=== | ||
*Divide into four pcr tubes of 55ul with total amplicon of ~10ug | *Divide into four pcr tubes of 55ul with total amplicon of ~10ug |
Revision as of 21:02, 19 July 2014
Agi26k0gap Probe Production
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:2014-07-17 Agi26k0gapProductionPCR.JPG
EtOH Precipitation
- 6 15-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 30min
- 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: 138.8 ng/ul => 33.3 ug