Revision as of 02:37, 16 January 2015 by >Mzcai
CA12k_Nov2014_V7 Probe Production[edit]
Production PCR[edit]
Components
|
Volume (1X)
|
Volume (50X)
|
First round amplicon CA12k_Nov14_V7 (10nM) |
0.2 |
10
|
2X KAPA SYBG MM |
50 |
2500
|
AP1V7U (100uM) |
0.4 |
20
|
AP2V7 (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
File:011214 CA12kNov14 ProductionPCR V7.JPG
EtOH Precipitation[edit]
- 6 15-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 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[edit]
- Purified 6 tubes of 100ul in 6 columns following Qiagen protocol
- Eluted each column with 50ul and combined all into one 1.5ml
- Measured concentration with Nanodrop:
~290ul of V7 probes: 93.2 ng/ul => ~27 ug
Lambda Exonuclease Digestion[edit]
- Divide each into 3 pcr tubes of ~100ul with total amplicon of <10ug each
Components
|
Volume
|
Amplicon |
100
|
10X Lambda Exo Buffer |
12.5
|
Lambda Exonuclease |
12.5
|
Total |
125
|
- Incubated at 37C for 1hr
- Purified with 3 Zymo ssDNA/RNA columns
- Eluted with 40ul each
- Recombined and measured ssDNA with Nanodrop:
- 62.9ng/ul x 120ul = 7.5ug (56% yield)
Remove Amplification Adapters[edit]
- Save 2ul for gel check later
- Split into 2 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)
TBU Gel: Digestion Check[edit]
- Combine into 1 tube
- 2ul Sample + 2ul 2X Urea Loading Dye
- 0.5ul Low Mass Ladder + 1.5ul H2O + 2ul 2X Urea Loading Dye
File:2015-01-14 V7 Digest GelCheck.jpg
- Gel ran poorly for some reason but I don't have anymore undigested probe to compare to (unless I want to waste 5ul of 10nM 1st rnd amplicon) so I will just check the gel when I do size selection
- The band to cut out should be 150bp so almost halfway between 200bp and 100bp ladder bands
Zymo Column Purification[edit]
- Eluted 30ul each column (60ul total)
- Nanodrop
- 41.7ng/ul x 60ul = 2.5ug (oddly low again like last time)
- This time 33% yield which is better than 25% yield last time
PAGE Size Selection[edit]
Components
|
1X Volume
|
V7 Probes |
60
|
TBE-Urea Buffer 2X |
60
|
Components
|
Volume
|
Low Mass Ladder |
1
|
TBE-Urea Buffer 2X |
5
|
H2O |
4
|
Total |
10
|
File:2015-01-14 SizeSelect V7.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.
- Transfered 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 60 min at 37 C in incubator
- Centrifuged at 15,000 rpm for 3 min at RT
- Transfered the clear spnt. to 2 Nanosep columns 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 2 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 overnight
- Spun 2 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 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:2015-01-15 V7 GelQuant.jpg
- Only use ladder bands 100bp, 200bp, and 400bp to get trendline with better R^2
- 28 ng/ul => 28 ng/ul / (150nt*325Da/nt + 79Da) = 573nM (18ul)
How I calculated concentration