Revision as of 20:12, 5 June 2015 by >Mzcai
CA12k_Nov2014_V4 Probe Production
Production PCR
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
File:20150603 CA12kNov2014 V4 ProductionPCR.JPG
QIAEX-II Purification
protocol
- 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
- 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: 300ul
- Nanodrop quantification: 155.7 ng/ul -> 46.7 ug
Lambda Exonuclease Digestion
- Divide into 6 pcr tubes of 50ul with total amplicon of <10ug each
Components
|
Volume
|
Amplicon |
50
|
10X Lambda Exo Buffer |
10
|
Lambda Exonuclease |
10
|
H2O |
30
|
Total |
100
|
- Incubated at 37C for 1hr20min
- Purified with 6 Zymo ssDNA/RNA columns
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- 59.6ng/ul x 240ul = 14.3ug (61% yield)
Remove Amplification Adapters
USER
- Split into 4 PCR tubes and add 5ul USER
Components
|
Volume
|
SS-amplicon |
59
|
USER |
5
|
10X DpnII Buffer |
8
|
H2O |
8
|
Total |
80
|
- Incubate at 37C for 2.5 hours
DpnII
- Added 15ul of the following
Components
|
Volume
|
10X DpnII Buffer |
2
|
100uM RE-DpnII_V4 guide oligo |
5
|
H2O |
8
|
- Incubated at 94C for 2 min, then 37C for 3 min
- Added 5ul DpnII
- Incubated at 37C for overnight (~15hrs)
Zymo Column Purification
- Eluted 30ul each column (115ul total)
- Nanodrop
- 51.9ng/ul x 115ul = 6.2ug (43% yield)
PAGE Size Selection
Components
|
2X Volume
|
V4 Probes |
115
|
TBE-Urea Buffer 2X |
115
|
Components
|
2X Volume
|
Low Mass Ladder |
2
|
TBE-Urea Buffer 2X |
10
|
H2O |
8
|
Total |
20
|