Matt:LabNotes/2017-5-20: Difference between revisions
Jump to navigation
Jump to search
>Mzcai (Created page with "==Agi15k_Feb2017_V4 Probe Production== *Production of Padlock Probes (V4) Matt:LabNotes/2017-2-8 ===Notes from last time=== *Do 12 or 13 cycle...") |
>Mzcai mNo edit summary |
||
Line 28: | Line 28: | ||
|} | |} | ||
'''Program''' 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 13 -> 72C 2min -> 15C hold<br> | '''Program''' 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 13 -> 72C 2min -> 15C hold<br> | ||
===EtOH Precipitation=== | ===EtOH Precipitation=== | ||
Line 39: | Line 36: | ||
**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 10,000rpm at 4C for 30 min | *Centrifuged at 10,000rpm at 4C for 30 min | ||
*Discarded supernatant and added 800ul of cold 80% EtOH | *Discarded supernatant and added 800ul of cold 80% EtOH |
Revision as of 23:08, 20 May 2017
Agi15k_Feb2017_V4 Probe Production
- Production of Padlock Probes (V4) Matt:LabNotes/2017-2-8
Notes from last time
- Do 12 or 13 cycles Production PCR
- Experiment with 1hr vs 2hr Lambda digestion
- Think of way to maximize USER and DpnII digestion
Production PCR
- 25nM 1st round amplicons
- V4 master mix made in 2 5ml tubes
Components | Volume (1X) | Volume (100X) |
First round amplicon Agi15k_Feb2017_V4 (25nM) | 0.1 | 10 |
2X KAPA SYBG MM | 50 | 5000 |
AP1V4U (100uM) | 0.4 | 40 |
AP2V4 (100uM) | 0.4 | 40 |
H2O | 49 | 4910 |
Total | 100 | 10000 |
Program 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 13 -> 72C 2min -> 15C hold
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