Revision as of 18:10, 7 May 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:20150505 CA12kNov2014 V4 ProductionPCR.JPG
EtOH Precipitation
- 12 5-ml tubes (with 8 wells of PCR product each) for V4
- 800ul PCR product
- 2000ul 100% EtOH
- 2.7ul GlycoBlue
- 80ul 3M NaOAc pH 5.2-5.5
- Vortexed and put in -80C for 30min
- Centrifuged at 3000rpm at 4C for 30 min
- Discarded supernatant and added 800ul of cold 80% EtOH
- Transferred DNA pellet (with 1ml pipette tip) to 12 1.5mL tubes
- Centrifuged 1.5ml tubes at 14,000rpm for 5 min at 4C
- Discarded supernatant and air-dried for 30 min in hood
- Resuspended DNA with 100ul H2O
Qia Column Purification
- Purified 12 tubes of 100ul in 12 columns following Qiagen protocol
- Eluted each column with 50ul and combined all into one 1.5ml
- Measured concentration with Nanodrop:
~600ul of V4 probes: 59.8 ng/ul => ~35.9 ug
Lambda Exonuclease Digestion
- Divide into 6 pcr tubes of ~100ul with total amplicon of <10ug each
Components
|
Volume
|
Amplicon |
96
|
10X Lambda Exo Buffer |
12
|
Lambda Exonuclease |
12
|
Total |
120
|
- Incubated at 37C for 1hr
- Purified with 6 Zymo ssDNA/RNA columns
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- 50.4 ng/ul x 240ul = 12.1 ug (67% 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 20ul each column (80ul total)
- Nanodrop
- 63.6ng/ul x 80ul = 5.0ug (54% yield)