Matt:LabNotes/2014-7-17: Difference between revisions
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>Mzcai (Created page with "==Agi26k0gap Probe Production== ===Production PCR=== *Make Master Mix for 46 well (6 strips) *0 gap first round amplicon -> V6 primers {| {{table}} | align="center" style="b...") |
>Mzcai mNo edit summary |
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98C 30sec -> (98C 10sec -> 55C 20sec -> 72C 30sec) x 15 -> 72C 2min -> 15C hold | 98C 30sec -> (98C 10sec -> 55C 20sec -> 72C 30sec) x 15 -> 72C 2min -> 15C hold | ||
[[File: |650px]] | [[File:2014-07-17_Agi26k0gapProductionPCR.JPG|650px]] | ||
===EtOH Precipitation=== | ===EtOH Precipitation=== | ||
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*Vortexed and put in -80C for 1hr | *Vortexed and put in -80C for 1hr | ||
*Centrifuged at 3000rpm at 4C for | *Centrifuged at 3000rpm at 4C for overnight<!-- | ||
*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 |
Revision as of 01:32, 18 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 5-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 1hr
- Centrifuged at 3000rpm at 4C for overnight