Daniel:Notebook/ComboLock/2017-3-2: Difference between revisions
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>Djacobse |
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<li>Prepare 8.2X Master mix according to table below</li> | <li>Prepare 8.2X Master mix according to table below</li> | ||
<li>Add | |||
<li>Add sample | {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | ||
|- style="background-color:#8DB4E2;font-size:12pt;font-weight:bold" align="center" | |||
| width="160" height="30" | Reagent | |||
| width="85" | Single Rxn Vol | |||
| width="85" | 16.2X MM uL | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Template | |||
| align="right" align="center" valign="bottom" | 4 | |||
| align="right" align="center" valign="bottom" | 0 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | RCA Primer (10 uM) | |||
| align="right" align="center" valign="bottom" | 2.5 | |||
| align="right" align="center" valign="bottom" | 0 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | dNTP (1 mM) | |||
| align="right" align="center" valign="bottom" | 5 | |||
| align="right" align="center" valign="bottom" | 81 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | 10X Buffer | |||
| align="right" align="center" valign="bottom" | 2 | |||
| align="right" align="center" valign="bottom" | 32.4 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Phi29 | |||
| align="right" align="center" valign="bottom" | 1 | |||
| align="right" align="center" valign="bottom" | 0 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | BSA (10 mg/mL) | |||
| align="right" align="center" valign="bottom" | 0.4 | |||
| align="right" align="center" valign="bottom" | 6.48 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | nfH2O | |||
| align="right" align="center" valign="bottom" | 5.1 | |||
| align="right" align="center" valign="bottom" | 82.62 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | Total | |||
|style="font-weight:bold" align="right" align="center" valign="bottom" | 20 | |||
|style="font-weight:bold" align="right" align="center" valign="bottom" | 202.5 | |||
|} | |||
<li>Add 12.5 uL master mix to each tube</li> | |||
<li>Add 4 uL template according to table</li> | |||
<li>Add 2.5 uL correct primer according to table</li> | |||
<li>Incubate at 95 C for 5 minutes</li> | |||
<li>Lower to 55C for 15 minutes</li> | |||
<li>Lower to 30C for at least 2 minutes</li> | |||
<li>Add 1 uL Phi29 polymerase to each sample</li> | |||
<li>Incubate 3 hours at 30C</li> | |||
<li>Heat kill enzyme by incubating 10 minutes at 65C; Hold at 12C</li> | |||
</ol></ol> | |||
==Protocol-Part 4== | |||
<ol start="9"> | |||
<li>BglII Digestion</li> | |||
<ol type="A"> | |||
<li>Use samples 1 (Experiment) and 4 (No phosphate) for Bglii digest</li> | |||
<li>Make the following 4.2X master mix</li> | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#8DB4E2;font-size:12pt;font-weight:bold" align="center" | |||
| width="200" height="30" | Reagent | |||
| width="85" | Single Rxn Vol | |||
| width="85" | 8.2X MM uL | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | RCA Reaction Product | |||
| align="center" align="center" valign="bottom" | 5 | |||
| align="center" align="center" valign="bottom" | 0 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | Bglii Cutter Primer (10 uM) | |||
| align="center" align="center" valign="bottom" | 2 | |||
| align="center" align="center" valign="bottom" | 16.4 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | 10X Buffer 3.1 | |||
| align="center" align="center" valign="bottom" | 2 | |||
| align="center" align="center" valign="bottom" | 16.4 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | Bglii | |||
| align="center" align="center" valign="bottom" | 1 | |||
| align="center" align="center" valign="bottom" | 0 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | nfH2O | |||
| align="center" align="center" valign="bottom" | 10 | |||
| align="center" align="center" valign="bottom" | 82 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| 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" | 114.8 | |||
|} | |||
<li>Aliquot 14 uL master mix into new tubes</li> | |||
<li>Add 5 uL appropriate sample to each tube</li> | |||
<li>Use the following thermocycler program</li> | |||
<ol type="a"> | |||
<li>5 min 95C</li> | |||
<li>10 min 50C</li> | |||
<li>1 hr 37C; '''When this cycle starts add 1 uL Bglii'''</li> | |||
<li>Heat kill with 65C for 20 min</li></ol> | |||
</ol> | |||
<li>qPCR</li> | |||
<ol type="A"> | |||
<li>Make 25.2X qPCR master mix according to following recipe</li> | |||
<ol type="a"> | |||
<li>529.2 uL nfH2O</li> | |||
<li>630 uL SYBR Fast</li> | |||
<li>25.2 uL 10 uM AmpF</li> | |||
<li>25.2 uL 10 uM AmpR-Ind20</li> | |||
</ol> | |||
<li>Add 48 uL master mix to each well</li> | |||
<li>Add 2 uL sample according to plate layout</li> | |||
[[Image:PlateLayout-20170302-Padlock0601Test.png|600px]] | |||
<li>qPCR Cycles</li> | |||
<ol type="a"> | |||
<li>95C 3 min</li> | |||
<li>95C 3 sec</li> | |||
<li>55C 30 sec</li> | |||
<li>72C 20 sec</li> | |||
<li>plate read</li> | |||
<li>goto b x30</li> | |||
<li>72C 2 min</li> | |||
<li>16C hold</li> | |||
</ol></ol></ol> | |||
qPCR for post-Bglii digestion and gel images for all data continued [[Daniel:Notebook/ComboLock/2017-3-3|tomorrow]] | |||
[[Category:ComboLock]] [[Category:20170301]] | [[Category:ComboLock]] [[Category:20170301]] |
Latest revision as of 18:25, 6 March 2017
Padlock0601 Test (Started Yesterday)[edit]
Protocol-Part 3[edit]
- Exonuclease Digestion
- Mix 15 uL Exo I (20U/uL) and 3 uL ExoIII (100U/uL)
- Add 2 uL to each sample, mix by swirling pipette tip
- Incubate for 1.5 hours at 37C
- Heat kill by incubating for 5 min at 95C
- Column Purification-Qiaquick
- Add 150 uL (5X) PB (binding buffer) to 30 uL sample
- Load sample onto column and spin for 1 minute at 14000 rpm; discard flow through
- Add 750 uL PE (wash buffer) to column and spin for 1 minute at 14000 rpm; discard flow through;
- Repeat wash step;
- Dry spin column for 1 minute at 14000 rpm; discard flow through
- Let stand with cover open in fume hood for ~5 minutes
- Transfer column to a new 1.5 mL eppendorf tube
- Elute with 30 uL EB
- Let stand 1 minute
- Spin for 1 minute at 14000 rpm
- Rolling Circle Amplification
- Samples will be aliquoted under following rules
- Prepare 8.2X Master mix according to table below
- Add 12.5 uL master mix to each tube
- Add 4 uL template according to table
- Add 2.5 uL correct primer according to table
- Incubate at 95 C for 5 minutes
- Lower to 55C for 15 minutes
- Lower to 30C for at least 2 minutes
- Add 1 uL Phi29 polymerase to each sample
- Incubate 3 hours at 30C
- Heat kill enzyme by incubating 10 minutes at 65C; Hold at 12C
Samples | Condition |
Sample X (1-4, AB) | Unpurified |
Sample Y (1-4, AB) | Purified |
A Samples (1-4) | Normal Primer |
B Samples (1-4) | Phosphorothiorate Primer |
Reagent | Single Rxn Vol | 16.2X MM uL |
Template | 4 | 0 |
RCA Primer (10 uM) | 2.5 | 0 |
dNTP (1 mM) | 5 | 81 |
10X Buffer | 2 | 32.4 |
Phi29 | 1 | 0 |
BSA (10 mg/mL) | 0.4 | 6.48 |
nfH2O | 5.1 | 82.62 |
Total | 20 | 202.5 |
Protocol-Part 4[edit]
- BglII Digestion
- Use samples 1 (Experiment) and 4 (No phosphate) for Bglii digest
- Make the following 4.2X master mix
- Aliquot 14 uL master mix into new tubes
- Add 5 uL appropriate sample to each tube
- Use the following thermocycler program
- 5 min 95C
- 10 min 50C
- 1 hr 37C; When this cycle starts add 1 uL Bglii
- Heat kill with 65C for 20 min
- qPCR
- Make 25.2X qPCR master mix according to following recipe
- 529.2 uL nfH2O
- 630 uL SYBR Fast
- 25.2 uL 10 uM AmpF
- 25.2 uL 10 uM AmpR-Ind20
- Add 48 uL master mix to each well
- Add 2 uL sample according to plate layout File:PlateLayout-20170302-Padlock0601Test.png
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x30
- 72C 2 min
- 16C hold
Reagent | Single Rxn Vol | 8.2X MM uL |
RCA Reaction Product | 5 | 0 |
Bglii Cutter Primer (10 uM) | 2 | 16.4 |
10X Buffer 3.1 | 2 | 16.4 |
Bglii | 1 | 0 |
nfH2O | 10 | 82 |
Total | 20 | 114.8 |
qPCR for post-Bglii digestion and gel images for all data continued tomorrow