Daniel:Notebook/ComboLock/2017-3-25: Difference between revisions
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<li>Bglii digest</li> | <li>Bglii digest</li> | ||
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<li>Make the following reactions; '''Do not add Bglii yet'''</li> | <li>Make the following reactions (; '''Do not add Bglii yet'''</li> | ||
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|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | | | height="15" valign="bottom" | Circular Product (1X) | ||
| align="center" align="center" valign="bottom" | 4 | | align="center" align="center" valign="bottom" | 4 | ||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| height="15" valign="bottom" | Bglii Cutter Primer ( | | height="15" valign="bottom" | Bglii Cutter Primer (100 uM) | ||
| align="center" align="center" valign="bottom" | 5 | | align="center" align="center" valign="bottom" | 5 | ||
Revision as of 23:40, 25 March 2017
RCA Test
This experiment is to test the effectiveness of RCA so that I can use the most efficient method of amplification for the cell-based experiments. This builds on the T4 experiments and the circular product production. I will be using that circular product to analyze the effectiveness of RCA.
Linear Product Production (Continued from yesterday)
- TBE gel
- Mix 64 uL TBE and 16 uL 6X dye
- Aliquot 10 uL on to parafilm per sample/ladder
- Add 2 uL reaction, 1 uL (10 uM) controls, and 1.5 uL ladder to appropriate aliquot File:GelLanes-20170325-LinearProduct.png
- Add 10 uL mix to gel lanes
- Run gel for 24 minutes at 230V
- Stain with 2 uL SYBR gold for 3 minutes
- Rinse and image in gel doc
File:2017-03-25-LinearProductProduction.png
Still too much circular product present. I'll try one more time to make it linear.
Linear Product Production
This time we'll start with the circular product and chop it.
- Bglii digest
- Make the following reactions (; Do not add Bglii yet
- Use the following thermocycler program
- 5 min 95C
- Ramp to 50C at 0.2C/s
- 10 min 50C
- 4 hr 37C; When this cycle starts add 1 uL Bglii
- Heat kill with 65C for 20 min
- Rolling Circle Amplification
- Prepare master mix according to table below
- Add 16.5 uL master mix to each tube
- Add 2.5 uL 10 uM RCA primer and to new reaction tubes according to following rules
- "A" samples get LLRC
- "B" samples get LLRC-v03 (Mono-phosphorothiorate)
- "B" samples get LLRC-v04 (Tri-phosphorothiorate)
- Heat reactions up to 95C for 5 min
- Cool to 55C and incubate for 15 minutes
- Cool to 30C and add enzyme
- Incubate at 30C for length of time according to table below
- Incubate at 65C for 10 minutes;Hold at 10C until next step
Reagent | Single Rxn Vol |
Circular Product (1X) | 4 |
Bglii Cutter Primer (100 uM) | 5 |
10X Buffer 3.1 | 2 |
Bglii | 1 |
nfH2O | 8 |
Total | 20 |
RCA Protocol
Reagent | Single Rxn (uL) | Master Mix X+ (6.2X) | Master Mix X- (6.2X) | Master Mix Y+ (3.2X) | Master Mix Y- (3.2X) | Master Mix Z+ (3.2X) | Master Mix Z- (3.2X) | Master Mix P+ (3.2X) | Master Mix P- (3.2X) | Master Mix Q+ (3.2X) | Master Mix Q- (3.2X) |
Enzyme | NA | NEB | NEB | Thermo | Thermo | Epi (1:10) | Epi (1:10) | NEB | NEB | NEB | NEB |
Circular Product (1X) | 4 | 24.8 | 0 | 12.8 | 0 | 12.8 | 0 | 12.8 | 0 | 12.8 | 0 |
Linear Product (1X) | 4 | 0 | 24.8 | 0 | 12.8 | 0 | 12.8 | 0 | 12.8 | 0 | 12.8 |
RCA Primer (10 uM) | 2.5 | 15.5 | 15.5 | 8 | 8 | 8 | 8 | 8 | 8 | 8 | 8 |
dNTP (1 mM) | 2 | 12.4 | 12.4 | 6.4 | 6.4 | 6.4 | 6.4 | 6.4 | 6.4 | 6.4 | 6.4 |
10X Buffer | 2 | 12.4 | 12.4 | 6.4 | 6.4 | 6.4 | 6.4 | 6.4 | 6.4 | 6.4 | 6.4 |
Phi29 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
BSA (10 mg/mL) | 0.4 | 2.48 | 2.48 | 0 | 0 | 0 | 0 | 1.28 | 1.28 | 1.28 | 1.28 |
nfH2O | 8.1 | 50.22 | 50.22 | 25.92 | 25.92 | 25.92 | 25.92 | 25.92 | 25.92 | 25.92 | 25.92 |
Total | 20 | 117.8 | 117.8 | 59.52 | 59.52 | 59.52 | 59.52 | 60.8 | 60.8 | 60.8 | 60.8 |
Sample | Enzyme | Incubation Time (hrs) |
X (ABC) | NEB | 3 |
Y (ABC) | Thermo | 3 |
Z (ABC) | Epi (1:10) | 3 |
P (ABC) | NEB | 6 |
Q (ABC) | NEB | 12 |