Revision as of 19:42, 5 June 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:20150603 CA12kNov2014 V4 ProductionPCR.JPG
QIAEX-II Purification
protocol
- Divide into 8 5mL tubes of 1200ul each
- Add 3600ul (3X volume) of Buffer QX1
- Vortex the QIAEXII for 30sec
- Add 30ul QIAEXII (30ul for every 10ug)
- Incubate at 50C while mixing for 10min
- Centrifuge sample for 30sec
- Remove supernatant with a pipette
- Wash the pellet with 500ul Buffer QX1
- Resuspend the pellet by vortexing
- Transfer to 1.5ml tubes
- Centrifuge sample for 30sec and remove supernatant again with pipette
- Wash the pellet twice with 500ul Buffer PE
- Air-dry the pellet for 30min until the pellet turns white
- To elute, resuspend with 20ul H2O and vortexing
- Incubate at RT for 5min
- Centrifuge for 30sec and pipette out supernatant with sample
- Repeat 20ul H2O resuspension and incubation to improve yield 10-15%
- Total volume: 300ul
- Nanodrop quantification: 155.7 ng/ul -> 46.7 ug
Lambda Exonuclease Digestion
- Divide into 6 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 1hr20min
- Purified with 6 Zymo ssDNA/RNA columns
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- 59.6ng/ul x 240ul = 14.3ug (61% 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
- 51.9ng/ul x 115ul = 6.2ug (43% yield)
PAGE Size Selection
Components
|
3X Volume
|
V4 Probes |
115
|
TBE-Urea Buffer 2X |
115
|
Components
|
3X Volume
|
Low Mass Ladder |
3
|
TBE-Urea Buffer 2X |
15
|
H2O |
12
|
Total |
30
|
File:2015-02-04 CA12kNov14 SizeSelect V4 Gel1.jpg
File:2015-02-04 CA12kNov14 SizeSelect V4 Gel2.jpg
File:2015-02-04 CA12kNov14 SizeSelect V4 Gel3.jpg
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 2.0 mL tube below with pipette tip.
- Added 900 ul of 1X TE buffer
- Vortexed for 120min at 37 C in incubator
- Centrifuged at 15,000 rpm for 3 min at RT
- Transferred the clear spnt. to 6 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 6 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 2 tubes at -80C for 20min
- Spun 6 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 10ul and combine
TBU Gel Quantification
- Dilute 2ul to 10ul with H2O
- Add 10ul 2X TBE-Urea Buffer
- Load wells with 2, 4, 6, and 8 ul
- Load Low Mass DNA ladders in 0.5, 1, and 2 ul
- Ran at 200 V for 35min
'
|
0.5 ladder
|
1 ladder
|
2 ul
|
4 ul
|
6 ul
|
8 ul
|
2 ladder
|
DNA volume |
0.5 |
1 |
0.1 |
0.2 |
0.3 |
0.4 |
2
|
H2O |
1.5 |
1 |
0.9 |
1.8 |
2.7 |
3.6 |
0
|
2X Buffer |
2 |
2 |
1 |
2 |
3 |
4 |
2
|
Total |
4 |
4 |
2 |
4 |
6 |
8 |
4
|
|
File:2015-02-05 CA12kNov14 V4 GelQuant.jpg
- Only use ladder bands 100bp, 200bp, to get trendline with better R^2
- 40ng/ul => 40ng/ul / (150nt*325Da/nt + 79Da) = 819nM (58ul)
How I calculated concentration
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