Revision as of 22:38, 10 February 2015 by >Mzcai
CA12k_Nov2014_V7 Probe Production[edit]
Production PCR[edit]
Components
|
Volume (1X)
|
Volume (100X)
|
First round amplicon CA12k_Nov14_V7 (10nM) |
0.2 |
20
|
2X KAPA SYBG MM |
50 |
5000
|
AP1V7U (100uM) |
0.4 |
40
|
AP2V7 (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:020515 CA12kNov14 ProductionPCR V7.JPG
EtOH Precipitation[edit]
- 12 5-ml tubes (with 8 wells of PCR product each) 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 90min
- 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[edit]
- 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:
~590ul of V7 probes: 107.3ng/ul => 63.3ug
Lambda Exonuclease Digestion[edit]
- Divide into 6 pcr tubes of ~100ul with total amplicon of ~10ug each
Components
|
Volume
|
Amplicon |
98
|
10X Lambda Exo Buffer |
12
|
Lambda Exonuclease |
12
|
Total |
122
|
- Incubated at 37C for 1hr
- Purified with 6 Zymo ssDNA/RNA columns
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- 78.1ng/ul x 240ul = 18.7ug (59% yield)
Remove Amplification Adapters[edit]
- 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
- Added 15ul of the following
Components
|
Volume
|
10X DpnII Buffer |
2
|
100uM RE-DpnII_V7 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[edit]
- Eluted 30ul each column
- Nanodrop
- 46.6ng/ul x 120ul = 5.6ug (30% yield)
PAGE Size Selection[edit]
Components
|
2X Volume
|
V7 Probes |
120
|
TBE-Urea Buffer 2X |
120
|
Components
|
2X Volume
|
Low Mass Ladder |
2
|
TBE-Urea Buffer 2X |
10
|
H2O |
8
|
Total |
20
|
File:2015-02-09 CA12kNov14 SizeSelect V7 Gel1.jpg
File:2015-02-09 CA12kNov14 SizeSelect V7 Gel2.jpg
EtOH Precipitation[edit]
- 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 60min at 37 C in incubator
- Centrifuged at 15,000 rpm for 3 min at RT
- Transferred the clear spnt. to 4 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 4 fresh 1.5 mL tube (~440ul per tube)
- Precipitated with 1000ul of 100% EtOH, 1.4ul of Glycoblue, 44ul of 3M NaoAc pH 5.2
- Vortexed and placed the 4 tubes at -80C for 30min
- Spun 4 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[edit]
- 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:2015-02-10 CA12kNov14 V7 GelQuant.jpg
- Only use ladder bands 100bp, 200bp, and 400bp to get trendline with better R^2
- 42ng/ul => 42ng/ul / (150nt*325Da/nt + 79Da) = 860nM (38ul)
How I calculated concentration