Daniel:Notebook/ComboLock/2017-4-29: Difference between revisions
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|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | | | height="15" valign="bottom" | p12RC*** (10 uM) | ||
| align="center" align="center" valign="bottom" | 2 | | align="center" align="center" valign="bottom" | 0.2 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 2.04 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 1.24 | ||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| height="15" valign="bottom" | | | height="15" valign="bottom" | RCA Primer LLRC*** (10 uM) | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 0.2 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 2.04 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 1.24 | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | dNTP (10 mM) | |||
| align="center" align="center" valign="bottom" | 0.5 | |||
| align="center" align="center" valign="bottom" | 5.1 | |||
| align="center" align="center" valign="bottom" | 3.1 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | 10X Buffer | | height="15" valign="bottom" | 10X Buffer | ||
| align="center" align="center" valign="bottom" | 2 | | align="center" align="center" valign="bottom" | 2 | ||
Line 54: | Line 60: | ||
| align="center" align="center" valign="bottom" | 12.4 | | align="center" align="center" valign="bottom" | 12.4 | ||
|- style=" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | Phi29 | | height="15" valign="bottom" | Phi29 | ||
| align="center" align="center" valign="bottom" | 1 | | align="center" align="center" valign="bottom" | 1 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 0 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 0 | ||
|- style="font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| height="15" valign="bottom" | BSA (10 mg/mL) | | height="15" valign="bottom" | BSA (10 mg/mL) | ||
| align="center" align="center" valign="bottom" | 0.4 | | align="center" align="center" valign="bottom" | 0.4 | ||
Line 66: | Line 72: | ||
| align="center" align="center" valign="bottom" | 0 | | align="center" align="center" valign="bottom" | 0 | ||
|- style=" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | nfH2O | | height="15" valign="bottom" | nfH2O | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 11.7 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 119.34 | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 75.02 | ||
|- style="font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| height="15" valign="bottom" | Total | | height="15" valign="bottom" | Total | ||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 20 | |style="font-weight:bold" align="center" align="center" valign="bottom" | 20 | ||
|style="font-weight:bold" align="center" align="center" valign="bottom" | | |style="font-weight:bold" align="center" align="center" valign="bottom" | 153 | ||
|style="font-weight:bold" align="center" align="center" valign="bottom" | | |style="font-weight:bold" align="center" align="center" valign="bottom" | 93 | ||
|} | |} |
Revision as of 20:21, 29 April 2017
Production Run (Started Yesterday)
Protocol-Part 4
- Exonuclease Digestion
- Mix 17.5 uL Exo I (20U/uL) and 3.5 uL ExoIII (100U/uL)
- Add 2 uL to each sample, mix by swirling pipette tip
- Incubate for 2 hours at 37C
- Heat kill by incubating for 5 min at 95C
- Rolling Circle Amplification
- Prepare master mix according to table below
- Add 15 uL master mix to each tube
- Samples "A-C" get + master mixes; samples "D-F" get - master mixes
- Add 1 uM RCA primer and to new reaction tubes according to following rules
- "A,D" samples get 2 uL each LLRC & primer12RC
- "B,E" samples get 2 uL each LLRC*** & primer12RC
- "C,F" samples get 4 uL LLRC***
- Heat reactions up to 95C for 5 min
- Cool to 55C and incubate for 15 minutes
- Cool to 30C and add enzyme
- Incubate for 3 hours at 30C
- Incubate for 65 minutes at 20C to denature
- qPCR
- Make qPCR master mixes according to following recipe
Reagent | Single Rxn (uL) | Master Mix X (10.2X) | Master Mix Y (6.2X) |
Enzyme | NA | NEB | Thermo |
Sample | 4 | 0 | 0 |
p12RC*** (10 uM) | 0.2 | 2.04 | 1.24 |
RCA Primer LLRC*** (10 uM) | 0.2 | 2.04 | 1.24 |
dNTP (10 mM) | 0.5 | 5.1 | 3.1 |
10X Buffer | 2 | 20.4 | 12.4 |
Phi29 | 1 | 0 | 0 |
BSA (10 mg/mL) | 0.4 | 4.08 | 0 |
nfH2O | 11.7 | 119.34 | 75.02 |
Total | 20 | 153 | 93 |