Daniel:Notebook/ComboLock/2016-12-16: Difference between revisions
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>Djacobse |
>Djacobse |
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=Overnight Capture (Started [[Daniel:Notebook/ComboLock/2016-12-15|Yesterday]]= | =Overnight Capture (Started [[Daniel:Notebook/ComboLock/2016-12-15|Yesterday]])= | ||
[[Daniel:Notebook/ComboLock|Back to Calendar]] | [[Daniel:Notebook/ComboLock|Back to Calendar]] | ||
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<li>Add 15 uL master mix to each tube</li> | <li>Add 15 uL master mix to each tube</li> | ||
<li>Add sample to new reaction tubes according to following table; '''use template from both supernatant only and beads'''</li> | <li>Add sample to new reaction tubes according to following table; '''use template from both supernatant only and beads'''</li> | ||
{| 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" | |||
| width="180" height="32" | Reagent | |||
| width="65" | uL Added | |||
| width="80" | Master Mix (7X) | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Template | |||
| align="center" align="center" valign="bottom" | 5 | |||
| align="center" valign="bottom" | NA | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | RCA Primer (100 uM) | |||
| align="center" align="center" valign="bottom" | 2.5 | |||
| align="center" align="center" valign="bottom" | 17.5 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | dNTP (1 mM) | |||
| align="center" align="center" valign="bottom" | 0.8 | |||
| align="center" align="center" valign="bottom" | 5.6 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | 10X Buffer | |||
| align="center" align="center" valign="bottom" | 2 | |||
| align="center" align="center" valign="bottom" | 14 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Phi29 | |||
| align="center" align="center" valign="bottom" | 1 | |||
| align="center" align="center" valign="bottom" | 7 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | nfH2O | |||
| align="center" align="center" valign="bottom" | 8.7 | |||
| align="center" align="center" valign="bottom" | 60.9 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Total | |||
|style="font-weight:bold" align="center" align="center" valign="bottom" | 20 | |||
| align="center" align="center" valign="bottom" | 105 | |||
|} | |||
<li>Incubate at 37C for 3 hours</li> | |||
<li>Incubate at 65C for 10 minutes;Hold at 10C until next step</li></ol> | |||
<li>qPCR</li> | |||
<ol type="A"> | |||
<li>Make master mix according to following recipe</li> | |||
<ol type="a"> | |||
<li>151.2 uL nfH2O</li> | |||
<li>180 uL SYBR Fast</li> | |||
<li>7.2 uL 10 uM AmpF</li> | |||
</ol> | |||
<li>Add 47 uL master mix to each well</li> | |||
<li>Add 2 uL sample and 1 uL appropriate Reverse Primer (1 uM) according to table</li> | |||
{| 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> | ||
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|} | |} | ||
</ol> | </ol> | ||
=Version 2 Oligos= | |||
[[Category:ComboLock]] [[Category:20161215]] |
Revision as of 17:36, 16 December 2016
Overnight Capture (Started Yesterday)
Protocol
- Circularization
- Prepare 7X uL Phusion mix
- Add 20 uL Phusion mix to 20 uL of reaction
- Incubate at 60C for 2 hours
- Exonuclease Digestion
- Mix 12.5 uL Exo I (20U/uL) and 2.5 uL ExoIII (100U/uL)
- Add 2 uL to each sample, mix by swirling pipette tip
- Incubate for 1 hour at 37C
- Heat kill by incubating for 5 min at 95C
- Rolling Circle Amplification
- Prepare 7X Master mix according to table below
- Add 15 uL master mix to each tube
- Add sample to new reaction tubes according to following table; use template from both supernatant only and beads
- Incubate at 37C for 3 hours
- Incubate at 65C for 10 minutes;Hold at 10C until next step
- qPCR
- Make master mix according to following recipe
- 151.2 uL nfH2O
- 180 uL SYBR Fast
- 7.2 uL 10 uM AmpF
- Add 47 uL master mix to each well
- Add 2 uL sample and 1 uL appropriate Reverse Primer (1 uM) according to table
Reagent | Stock Conc | Final Amount | 1x Vol (uL) | MM Vol (7x) (uL) |
NAD+ | 5 mM | 40 nmol | 8 | 56 |
dNTP | 1 mM | 600 pmol | 0.6 | 4.2 |
Betaine | 5 M | 15 umol | 3 | 21 |
10X AmpLigase Buffer | 10X | 1X | 2 | 14 |
Amp Ligase | 5 U/uL | 10 U | 2 | 14 |
Phusion HF DNA Polymerase | 2000 U/mL | 6.4U | 3.2 | 22.4 |
nf H2O | 1.2 | 8.4 | ||
Total | 20 | 140 |
Reagent | uL Added | Master Mix (7X) |
Template | 5 | NA |
RCA Primer (100 uM) | 2.5 | 17.5 |
dNTP (1 mM) | 0.8 | 5.6 |
10X Buffer | 2 | 14 |
Phi29 | 1 | 7 |
nfH2O | 8.7 | 60.9 |
Total | 20 | 105 |
Sample | Condition | Fraction | Lane | AmpR Index | Sample Vol (uL) | 2X Kapa SYBR qPCR MM | 10 uM Forward Primer | 10 uM Reverse Primer | H2O | Total Volume (uL) |
Sample 1A | Sample | Supernatant | A1 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1B | Sample | Supernatant | A2 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2A | No Template | Supernatant | A3 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2B | No Template | Supernatant | A4 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3A | No C Probe | Supernatant | A5 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3B | No C Probe | Supernatant | A6 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1A | Sample | Beads | A1 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1B | Sample | Beads | A2 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2A | No Template | Beads | A3 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2B | No Template | Beads | A4 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3A | No C Probe | Beads | A5 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3B | No C Probe | Beads | A6 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
NTC | qPCR Primers Only | A7 | 23 | 2 | 25 | 1 | 1 | 23 | 50 |