Matt:LabNotes/2015-6-3: Difference between revisions
Jump to navigation
Jump to search
>Mzcai |
>Mzcai |
||
Line 25: | Line 25: | ||
===QIAEX-II Purification=== | ===QIAEX-II Purification=== | ||
[http://www.qiagen.com/us/resources/download.aspx?id=13d33145-9f64-426a-a43b-394211d8cf2b&lang=en protocol] | |||
*Divide into 8 5mL tubes of 1200ul each | *Divide into 8 5mL tubes of 1200ul each | ||
*Add 3600ul (3X volume) of Buffer QX1 | *Add 3600ul (3X volume) of Buffer QX1 |
Revision as of 02:46, 4 June 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
QIAEX-II Purification
- Divide into 8 5mL tubes of 1200ul each
- Add 3600ul (3X volume) of Buffer QX1
- Vortex the QIAEXII for 30sec
- Add 30ul QIAEXII (30ul for every 10ug)
- Incubate at 50C while mixing for 10min
- Color should be yellow
- Centrifuge sample for 30sec
- Remove supernatant with a pipette
- Wash the pellet with 500ul Buffer QX1
- Resuspend the pellet by vortexing
- Transfer to 1.5ml tubes
- Centrifuge sample for 30sec and remove supernatant again with pipette
- Wash the pellet twice with 500ul Buffer PE
- Air-dry the pellet for 30min until the pellet turns white
- To elute, resuspend with 20ul H2O and vortexing
- Incubate at RT for 5min
- Centrifuge for 30sec and pipette out supernatant with sample
- Repeat 20ul H2O resuspension and incubation to improve yield 10-15%
- Total volume: 320ul
- Nanodrop quantification: