Revision as of 23:49, 15 November 2016 by >Mzcai
CA12k_Oct2016 V4 Probe Preparation
Production PCR
Components
|
Volume (1X)
|
Volume (50X)
|
First round amplicon CA12k_Oct2016_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
|
- Did one plate - 96 wells 100ul each
Program 98C 1min -> (98C 30sec -> 55C 45sec -> 72C 45sec) x 14 -> 72C 2min -> 15C hold
450px
EtOH Precipitation
- 16 5-ml tubes each with
- 1,200ul PCR product
- 3000ul 100% EtOH
- 4ul GlycoBlue
- 120ul 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 16 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
- 16 columns elute 50ul each
- Nanodrop: 170.3 ng/uL x 800uL = 136.24ug
Lambda Exo
- Divide into 14 pcr tubes of 100ul (ideally should be <10ug each)
Components
|
Volume
|
Amplicon |
57
|
10X Lambda Exo Buffer |
10
|
Lambda Exonuclease |
10
|
H2O |
23
|
Total |
100
|
- Incubated at 37C for 1hr
- Purified with 14 Zymo ssDNA/RNA columns
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- 72.4ng/ul x 560ul = 40.54ug (60% yield)
Remove Amplification Adapters
USER
- Split into 9 PCR tubes and add 5ul USER
Components
|
Volume
|
SS-amplicon |
62.2
|
USER |
5
|
10X DpnII Buffer |
8
|
H2O |
4.8
|
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 (~12hrs)
Zymo Column Purification
- Eluted 20ul each column (180ul total)
- Nanodrop
- 80.1 ng/ul x 180ul = 14.4ug (36% yield lower than normal)
PAGE Size Selection
- Run 3 gels (normally should be 5 gels to keep < 3ug per gel)
- 200V for 40min
Components
|
2X Volume
|
V4 Probes |
180
|
TBE-Urea Buffer 2X |
180
|
Components
|
2X Volume
|
Low Mass Ladder |
3
|
TBE-Urea Buffer 2X |
15
|
H2O |
12
|
Total |
30
|
EtOH Precipitation
- Put cut out gel in 6 0.5mL tubes (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 450 ul of 1X TE buffer to each
- Vortexed for 60min at 37 C in incubator
- Centrifuged at 15,000 rpm for 3 min at RT
- Transferred the clear spnt. to 2 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 fresh 1.5 mL tube
- Precipitated in 6 1.5ml tubes with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 45ul of 3M NaoAc pH 5.2
- Vortexed and placed the 6 tubes at -80C for 30min
- Spun 6 tubes at 10,000rpm at 4C for 30min
- Discard supernatant and add 750ul 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 10ul and combine
Qubit ssDNA
7.79ng/ul x 60ul = 467.4ng
7.79ng/ul / (157 x 303.7Da/nt + 79Da) = 160nM