Daniel:Notebook/ComboLock/2017-7-7: Difference between revisions
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>Djacobse |
>Djacobse m (→Protocol) |
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(3 intermediate revisions by the same user not shown) | |||
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{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | ||
|- style="background-color:#92CDDC;font-size:12pt;font-weight:bold" align="center" | |- style="background-color:#92CDDC;font-size:12pt;font-weight:bold" align="center" | ||
| width=" | | width="230" height="30" | Reagent | ||
| width="95" | Single Rxn (uL) | | width="95" | Single Rxn (uL) | ||
| width="95" | Master Mix X (8.2X) | | width="95" | Master Mix X (8.2X) | ||
Line 30: | Line 30: | ||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| height="15" valign="bottom" | Sample | | height="15" valign="bottom" | Sample | ||
| align=" | | align="right" align="center" valign="bottom" | 4 | ||
| align=" | | align="right" align="center" valign="bottom" | 0 | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | p12RC*** (10 uM) | | height="15" valign="bottom" | p12RC*** (10 uM) | ||
| align=" | | align="right" align="center" valign="bottom" | 0.2 | ||
| align=" | | align="right" align="center" valign="bottom" | 1.64 | ||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| height="15" valign="bottom" | RCA Primer LLRC*** (10 uM) | | height="15" valign="bottom" | RCA Primer LLRC*** (10 uM) | ||
| align=" | | align="right" align="center" valign="bottom" | 0.2 | ||
| align=" | | align="right" align="center" valign="bottom" | 1.64 | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | dNTP (10 mM) | | height="15" valign="bottom" | dNTP (10 mM) | ||
| align=" | | align="right" align="center" valign="bottom" | 0.5 | ||
| align=" | | align="right" align="center" valign="bottom" | 4.1 | ||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| height="15" valign="bottom" | 10X Buffer | | height="15" valign="bottom" | 10X Buffer | ||
| align=" | | align="right" align="center" valign="bottom" | 2 | ||
| align=" | | align="right" align="center" valign="bottom" | 16.4 | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | Phi29 | | height="15" valign="bottom" | Phi29 | ||
| align=" | | align="right" align="center" valign="bottom" | 1 | ||
| align=" | | align="right" align="center" valign="bottom" | 0 | ||
|- style="background-color:#BFBFBF;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=" | | align="right" align="center" valign="bottom" | 0.4 | ||
| align=" | | align="right" align="center" valign="bottom" | 3.28 | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | nfH2O | | height="15" valign="bottom" | nfH2O | ||
| align=" | | align="right" align="center" valign="bottom" | 11.7 | ||
| align=" | | align="right" align="center" valign="bottom" | 95.94 | ||
|- style="background-color:#BFBFBF;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=" | |style="font-weight:bold" align="right" align="center" valign="bottom" | 20 | ||
|style="font-weight:bold" align=" | |style="font-weight:bold" align="right" align="center" valign="bottom" | 123 | ||
|} | |} | ||
Line 89: | Line 89: | ||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | ||
|- style="font-size:12pt;font-weight:bold" align="center" | |- style="font-size:12pt;font-weight:bold" align="center" | ||
|style="background-color:#8DB4E2" width=" | |style="background-color:#8DB4E2" width="190" height="30" | Reagent | ||
|style="background-color:#8DB4E2" width="95" | Single Rxn Vol | |style="background-color:#8DB4E2" width="95" | Single Rxn Vol | ||
|style="background-color:#95B3D7" width="95" | Master Mix ( | |style="background-color:#95B3D7" width="95" | Master Mix (8.2X) | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | RCA Rxn | | height="15" valign="bottom" | RCA Rxn | ||
| align=" | | align="right" align="center" valign="bottom" | 7 | ||
| align=" | | align="right" align="center" valign="bottom" | 0 | ||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| height="15" valign="bottom" | 10X Buffer 3.1 | | height="15" valign="bottom" | 10X Buffer 3.1 | ||
| align=" | | align="right" align="center" valign="bottom" | 2 | ||
| align=" | | align="right" align="center" valign="bottom" | 16.4 | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | Bglii | | height="15" valign="bottom" | Bglii | ||
| align=" | | align="right" align="center" valign="bottom" | 1 | ||
| align=" | | align="right" align="center" valign="bottom" | 0 | ||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| height="15" valign="bottom" | nfH2O | | height="15" valign="bottom" | nfH2O | ||
| align=" | | align="right" align="center" valign="bottom" | 10 | ||
| align=" | | align="right" align="center" valign="bottom" | 82 | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | Total | | height="15" valign="bottom" | Total | ||
|style="font-weight:bold" align=" | |style="font-weight:bold" align="right" align="center" valign="bottom" | 20 | ||
|style="font-weight:bold" align=" | |style="font-weight:bold" align="right" align="center" valign="bottom" | 98.4 | ||
|} | |} | ||
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<li>Hold at 12C</ol> | <li>Hold at 12C</ol> | ||
</ol> | </ol> | ||
Continued [[Daniel:Notebook/ComboLock/2017-7-8|tomorrow]] | |||
[[Category:ComboLock]] [[Category:20170706]] | [[Category:ComboLock]] [[Category:20170706]] |
Latest revision as of 23:27, 7 July 2017
Oligo+Protein Test (Started Yesterday)[edit]
Protocol[edit]
- 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
- Add 4 uL sample to appropriate tubes
- Heat reactions up to 95C for 5 min
- Cool to 55C and incubate for 15 minutes
- Cool to 30C and add 1 uL enzyme
- Incubate for 3 hours at 30C
- Incubate for 65 minutes at 20C to denature
- Bglii digestion
- Make the following reactions; Do not add Bglii yet
- Aliquot 12 uL master mix into new tube
- Add 7 uL appropriate sample
- Mix with vortexer, spin down, and add 1 uL Bglii
- Incubate 2 hr 37C
- Heat kill with 65C for 20 min
- Hold at 12C
Reagent | Single Rxn (uL) | Master Mix X (8.2X) |
Enzyme | NA | Thermo |
Sample | 4 | 0 |
p12RC*** (10 uM) | 0.2 | 1.64 |
RCA Primer LLRC*** (10 uM) | 0.2 | 1.64 |
dNTP (10 mM) | 0.5 | 4.1 |
10X Buffer | 2 | 16.4 |
Phi29 | 1 | 0 |
BSA (10 mg/mL) | 0.4 | 3.28 |
nfH2O | 11.7 | 95.94 |
Total | 20 | 123 |
Reagent | Single Rxn Vol | Master Mix (8.2X) |
RCA Rxn | 7 | 0 |
10X Buffer 3.1 | 2 | 16.4 |
Bglii | 1 | 0 |
nfH2O | 10 | 82 |
Total | 20 | 98.4 |
Continued tomorrow