Revision as of 18:17, 21 July 2015 by >Mzcai
CA12k_Nov2014_V4 Probe Production
Production PCR
Components
|
Volume (1X)
|
Volume (100X)
|
First round amplicon CA12k_Nov14_V7 (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
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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 overnight
- 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 6 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 into one 1.5ml
- Measured concentration with Nanodrop:
~618ul of V4 probes: 76.4 ng/ul => ~47.2 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 columns
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- 63.5ng/ul x 233ul = 14.8ug (63% 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
- 54.7ng/ul x 115ul = 6.3ug (43% yield)
PAGE Size Selection
Components
|
2X Volume
|
V4 Probes |
120
|
TBE-Urea Buffer 2X |
120
|
Components
|
3X Volume
|
Low Mass Ladder |
2
|
TBE-Urea Buffer 2X |
10
|
H2O |
8
|
Total |
20
|
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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.
- Transferred the gel remaining in 0.5 mL tube to 1.5 mL tube below with pipette tip.
- Added 900 ul of 1X TE buffer (SHOULD HAVE BEEN 450ul)
- Vortexed for overnight at 37 C in incubator
- Centrifuged at 15,000 rpm for 3 min at RT
- Transferred the clear spnt. to 8 Nanosep columns and centrifuged at 15,000 rpm for 3 min
- Transferred the lower layer to Nanosep again and centrifuged at 15,000 rpm for 3 min
- Transferred spnt to 8 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 30min
- Spun 8 tubes at 10,000rpm at 4C for 30min
- Discard supernatant and add 650ul cold 80% EtOH and spin at 14,000rpm at 4C for 5min
- Discard supernatant and let dry in hood for 10min
- Resuspend each tube with 5ul and combine
Qubit Quantification
- 10.9ng/ul
- 10.9 ng/ul / (150nt*325Da/nt + 79Da) = 239nM (38ul)