Revision as of 21:17, 20 January 2015 by >Mzcai
CA12k_Nov2014 V4 and V7 Probe Production
Production PCR
Components
|
Volume (1X)
|
Volume (50X)
|
First round amplicon CA12k_Nov14_V4 (10nM) |
0.2 |
10
|
2X KAPA SYBG MM |
50 |
2500
|
AP1V4U (100uM) |
0.4 |
20
|
AP2V4 (100uM) |
0.4 |
20
|
H2O |
49 |
2450
|
Total |
100 |
5000
|
Components
|
Volume (1X)
|
Volume (50X)
|
First round amplicon CA12k_Nov14_V7 (10nM) |
0.2 |
10
|
2X KAPA SYBG MM |
50 |
2500
|
AP1V7U (100uM) |
0.4 |
20
|
AP2V7 (100uM) |
0.4 |
20
|
H2O |
49 |
2450
|
Total |
100 |
5000
|
Program 98C 1min -> (98C 15sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold
[[File: | 800px]]
EtOH Precipitation
- 6 15-ml tubes (with 8 wells of PCR product each) for V4 and 6 tubes for V7
- 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 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 5 min in hood
- Resuspended DNA with 50ul H2O
Qia Column Purification
- Purified in 6 columns following Qiagen protocol
- Eluted each column with 50ul and combined all V4 probes into one 1.5ml and all V7 probes into another
- Measured concentration with Nanodrop:
~150ul of V4 probes: 156.5 ng/ul => ~23.5 ug
~150ul of V7 probes: 163.6 ng/ul => ~24.5 ug
Lambda Exonuclease Digestion
- Divide each into 3 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 1hr
- Purified with 3 Zymo ssDNA/RNA columns each
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- V4: 77.0ng/ul x 120ul = 9.24ug (78.6% yield)
- V7: 61.0ng/ul x 120ul = 7.32ug (59.8% yield)
Remove Amplification Adapters
USER
- Split each set into 2 PCR tubes and add 5ul USER
Components
|
Volume
|
SS-amplicon |
60
|
USER |
5
|
H2O |
20
|
Total |
85
|
- Incubate at 37C for 2.5 hours
DpnII
- Added 15ul of the following
Components
|
Volume
|
10X DpnII Buffer |
10
|
100uM RE-DpnII guide oligo |
5
|
RE-DpnII guide oligo:
V4: RE DpnII V4
V7: RE DpnII V7
- Incubated at 94C for 2 min, then 37C for 3 min
- Added 5ul DpnII
- Incubated at 37C for overnight
TBU Gel: Digestion Check
- Combine into 1 tube for each set
- 2ul Digested Sample + 2ul 2X Urea Loading Dye
- 5ul Undigested V7 10nM 1st round amplicon) + 5ul 2X Urea Loading Dye
- 2X (0.5ul Low Mass Ladder + 1.5ul H2O + 2ul 2X Urea Loading Dye)
File:2015-01-20 V4V7 Digest GelCheck.jpg
- V7 Undigested (middle lane) is extremely faint but can see band around 200bp
- Not sure why V4 lane is squiggly but can see the 3 bands
- V7 lane shows only 2 bands but both are below the undigested V7 band