Revision as of 22:20, 21 September 2016 by >Mzcai
CA12k_Nov2014 V6 (RevComp of V4) Probe Production
Production PCR
Components
|
Volume (1X)
|
Volume (50X)
|
First round amplicon CA12k_Nov14_V6 (10nM) |
0.2 |
10
|
2X KAPA SYBG MM |
50 |
2500
|
AP1V6U (100uM) |
0.4 |
20
|
AP2V6 (100uM) |
0.4 |
20
|
H2O |
49 |
2450
|
Total |
100 |
5000
|
Program 98C 1min -> (98C 10sec -> 55C 30sec -> 72C 45sec) x 15 -> 72C 2min -> 15C hold
EtOH Precipitation
- 12 5-ml tubes (with 8 wells of PCR product each)
- 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 100ul H2O
Qia Column Purification
- Purified 12 tubes of 100ul in 12 columns following Qiagen protocol
- Eluted each column with 50ul and combined all V6 probes into one 1.5ml and all V8 probes into another
- Measured concentration with Nanodrop:
~600ul of V6 probes: 113.2 ng/ul => ~ 67.9 ug
Lambda Exonuclease Digestion
- Divide each into 8 pcr tubes of 75ul with total amplicon of <10ug each
Components
|
Volume
|
Amplicon |
75
|
10X Lambda Exo Buffer |
10
|
Lambda Exonuclease |
10
|
H2O |
5
|
Total |
100
|
- Incubated at 37C for 1hr
- Purified with 8 Zymo ssDNA/RNA columns
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- V6: 80.3ng/ul x 320ul = 25.7ug (75.6% yield)
Remove Amplification Adapters
USER
- Split each set into 5 PCR tubes and add 5ul USER
Components
|
Volume
|
SS-amplicon |
63
|
USER |
5
|
DpnII 10X |
8
|
H2O |
4
|
Total |
80
|
- Incubate at 37C for 3 hours
DpnII
- Added 15ul of the following
Components
|
Volume
|
10X DpnII Buffer |
2
|
100uM RE-DpnII_V6 guide oligo |
5
|
H2O |
8
|
- Incubated at 94C for 2 min, then 37C for 3 min
- Added 5ul DpnII
- Incubated at 37C for overnight (12 hrs)
Zymo Column Purification
- Eluted 20ul each column (100ul total)
- Nanodrop
- V6: 118.9ng/ul x 100ul = 11.9ug
PAGE Size Selection
Components
|
4X Volume
|
V6 Probes |
100
|
H2O |
20
|
TBE-Urea Buffer 2X |
120
|
Components
|
Volume
|
Low Mass Ladder |
4
|
TBE-Urea Buffer 2X |
20
|
H2O |
16
|
Total |
40
|
EtOH Precipitation
- Put cut out gel in 0.5mL tube (with holes at bottom and centrifuged the tube at 15,000 rpm, for 3 min at RT.)
- Transfered the gel remaining in 0.5 mL tube to 1.5 mL tube below with pipette tip.
- Added 450 ul of 1X TE buffer
- Vortexed for 60 min at 37 C in incubator
- Centrifuged at 15,000 rpm for 3 min at RT
- Transfered the clear spnt. to 8 Nanosep column and centrifuged at 15,000 rpm for 3 min
- Transfered the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min
- Transfered spnt to fresh 1.5 mL tube (~400ul per tube)
- Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 40ul of 3M NaoAc pH 5.2
- Vortexed and placed the 8 tubes at -80C for 30 min
- Spun 8 tubes at 10,000rpm at 4C for 30min
- Discard supernatant and add 650ul 80% EtOH and spin at 14,000rpm at 4C for 5min
- Discard supernatant and let dry in hood for 10min
- Resuspend each tube with 10ul and recombine (80ul total)
- Measure with qubit
- V6: 13.1 ng/ul => 13.1 ng/ul / (150nt*325Da/nt + 79Da) = 268.2nM (80ul)