Revision as of 06:10, 3 May 2015 by >Dinh
HOTSPOTS 460K
- Need more of Subset E. But to save time, I made more of A-E together in case I run out. Noi made one plate of Subsets A-F for me before she left, I still have a lot of subset F to use.
Current probes concentrations
Subset
|
Qubit (ng/uL)
|
A |
11.84
|
B |
15.4
|
C |
11.92
|
D |
11.76
|
E |
---
|
F |
5.12
|
Probes production (Day 1)
- Set up ~15 min + 30 min run / 96 reactions = 1.5 hr qPCR
- PCR cleanup with QIAEXII = 1 hr (would be faster if I can use an ultra-centrifuge to spin down 15 mL tubes).
- Used 1X2.0 mL tubes to prepare each reaction master mix:
Reagent Volume
KAPA 2X Sybr Fast 1,100 uL
Nuclease free water 1,100 uL
AP1V4 8.8 uL
AP2V4 8.8 uL
Template (10nM) 4.4 uL (template was prepared by Noi from starting probes mix).
95C 30s -> [ 95C 15s -> 55C 45s -> 60C 30s read plate -> 60C 10s]x20cycles -> 60C 1 min
- I used the CFX96 to perform qPCR. The incubation time at each temperature was increased to account for the overhead volume (>50uL).
- Subsets A,C,D appears to saturate at 14 cycles while B,E saturate at 15
- I used QIAEX II beads to purify the PCR reactions. Each 2,200 uL total reaction was split into 5x2.0 mL tube.
Used 20 uL of QIAEX II beads + 3X QX1 buffer.
Eluted each tube with 50 uL nuclease free water first, then added more water for serial dilution.
Subset
|
cycles
|
100 uL reactions
|
QIAEX II yield, Nanodrop (ng/uL)
|
QIAEX II volume (uL)
|
Total yield (ug)
|
A |
15 |
22 |
153.2 |
220 |
33.704
|
B |
15 |
22 |
128.4 |
220 |
28.248
|
C |
15 |
22 |
155.5 |
220 |
34.21
|
D |
15 |
22 |
154 |
230 |
35.42
|
E |
14 |
44 |
124.6 |
473 |
58.9358
|
- I expected 33 ug on average from each 22reactions and got about the right amount. Using QIAEX II beads saved time instead of performing ethanol precipitation and then Bioneer column purification.
Lambda Exo (Day 1)
Reagent Volume
dsDNA+water(~10ug) 125 uL
Lambda Exo buffer,10X 15 uL
Lambda Exo 10 uL
37C 1 hour -> 75C 10 min
- I set up 3x150 uL reactions for A-D, and 6x150uL reactions for E. I expected <20ug after digestion, so after pooling 3 reactions together in a 2.0 mL tube, I purified with 60 uL QIAEX II beads and 3X volumes QX1 buffer.
- Yield:
Subset
|
Input(ug)
|
QIAEX II yield, Nanodrop (ng/uL)
|
QIAEX II volume (uL)
|
Total LambdaExo/QIAEXII yield (ug)
|
%yield
|
A |
33.704 |
76.5 |
190 |
14.535 |
43%
|
B |
28.248 |
62.5 |
190 |
11.875 |
42%
|
C |
34.21 |
79.2 |
190 |
15.048 |
44%
|
D |
35.42 |
81.5 |
190 |
15.485 |
44%
|
E |
29.4679 |
67.1 |
190 |
12.749 |
43%
|
E |
29.4679 |
69.1 |
190 |
13.129 |
45%
|
- Check Lambda Exo efficiency by running PAGE TBE gel.
- LambdaExo digestion have good efficiency.
- QIAEX II can purify ssDNA
File:460K prep May2015 A-E LambdaExoDigestion.jpg
USER digestion (Day 1/Overnight)
- Set up time ~ 1 hr
- Set up 1x1.5 mL tube for each overnight reactions.
Reagent Volume
ssDNA+water(up to 15 ug) 280 uL
DpnII buffer (10X) 35 uL
USER (NEB) 35 uL
37C for overnight in thermomixer, 400 rpm
- Aliquot 2 uL of each for PAGE-verification.
DpnII digestion (Day 2)
- Set up time 30 minutes + 2 hours incubation + 30 minutes cleanup = 3 hr.
- To each USER 350 uL reaction, add 40 uL Guide-oligo (NN version), and add 5 uL DpnII buffer = 395 uL. Split into 5x0.2mL PCR tubes (79 ul each).
- Ramp to 37C, incubate at 37C for 5 minutes, to each PCR tube, add 1 uL of Dpn II (50U/uL).
- Incubate at 37C for 1 hour.
- Aliquot 2 uL of each (A-E) for PAGE-verification
- After 1 hour, add mixture of 8 uL water, 1 uL DpnII buffer, 1 uL of DpnII
- Incubate at 37C for 1 more hour.
- Aliquot 2 uL of each (A-E) for PAGE-verification
- Clean up 5x90 uL reactions using 2 ssDNA cleanup columns (Zymo). Use ssDNA column to remove all double strand DNA!
- Yield:
Agarose purification (Day 2)
- Use 13 wells gel to purify up to 6 ug at a time. Each gel runs for 50 minutes.
- Gel extraction and purification is 1 hr.
- So Up to 6 ug can be purified in 2 hours.