Daniel:Notebook/ComboLock/2017-1-30: Difference between revisions
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>Djacobse (Created page with "=RCA Test (Started Monday)= Back to Calendar ==Library Prep (Started [[Daniel:Notebook/ComboLock/2017-1...") |
>Djacobse |
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<li>Consolidate with samples from [[Daniel:Notebook/ComboLock/2017-1-26|Thursday 1-26]] | <li>Consolidate with samples from [[Daniel:Notebook/ComboLock/2017-1-26|Thursday 1-26]] | ||
</ol></ol> | </ol></ol> | ||
'''DEJ20160126-CLv3-RCA02''' | |||
'''Final Sample Matrix''' | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#8064A2;font-size:12pt;font-weight:bold" align="center" | |||
| width="160" height="30" | Sample | |||
| width="85" | Sample Amt | |||
| width="85" | Pre/Post RCA | |||
| width="85" | AmpR Index | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Sample 0 (AB) | |||
| align="center" valign="bottom" | 1 pmol | |||
| align="center" valign="bottom" | Pre | |||
| align="center" align="center" valign="bottom" | 21 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | Sample 0X (AB) | |||
| align="center" valign="bottom" | 1 pmol | |||
| align="center" valign="bottom" | Post | |||
| align="center" align="center" valign="bottom" | 25 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Sample 2X (AB) | |||
| align="center" valign="bottom" | 10 fmol | |||
| align="center" valign="bottom" | Post | |||
| align="center" align="center" valign="bottom" | 26 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | Sample 4X (AB) | |||
| align="center" valign="bottom" | 100 amol | |||
| align="center" valign="bottom" | Post | |||
| align="center" align="center" valign="bottom" | 27 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Sample 6C (AB) | |||
| align="center" valign="bottom" | 1 amol | |||
| align="center" valign="bottom" | Epi | |||
| align="center" align="center" valign="bottom" | 22 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | Sample 6D (AB) | |||
| align="center" valign="bottom" | 1 amol | |||
| align="center" valign="bottom" | Epi 1:10 | |||
| align="center" align="center" valign="bottom" | 23 | |||
|} | |||
=RCA Timing Test (Started [[Daniel:Notebook/ComboLock/2017-1-27|Friday]])= | |||
Because of the results from [[Daniel:Notebook/ComboLock/2017-1-28|Saturday]], I think I want to try Epicentre again for as long as possible. I'll do 12 hours with (as usual) one negative control to save up on epicentre enzyme. | |||
<ol> | |||
<li>Rolling Circle Amplification</li> | |||
<ol type="A"> | |||
<li>Prepare master mix according to table below</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="30" | Reagent | |||
| width="65" | uL Added | |||
| width="65" | Master Mix (4.2X) | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Template | |||
| align="center" align="center" valign="bottom" | 4 | |||
| align="center" valign="bottom" | NA | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | RCA Primer (10 uM) | |||
| align="center" align="center" valign="bottom" | 2.5 | |||
| align="center" align="center" valign="bottom" | 10.5 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | dNTP (1 mM) | |||
| align="center" align="center" valign="bottom" | 2 | |||
| align="center" align="center" valign="bottom" | 8.4 | |||
|- 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" | 8.4 | |||
|- style="font-size:12pt" | |||
| height="15" valign="bottom" | Phi29 | |||
| align="center" align="center" valign="bottom" | 1 | |||
| align="center" valign="bottom" | NA | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
| height="15" valign="bottom" | nfH2O | |||
| align="center" align="center" valign="bottom" | 8.5 | |||
| align="center" align="center" valign="bottom" | 35.7 | |||
|- style="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" | 63 | |||
|} | |||
<li>Add 14 uL master mix to each tube</li> | |||
<li>Add sample and to new reaction tubes according to following table</li> | |||
<li>Incubate at 30C for 12 hours</li> | |||
<li>Incubate at 65C for 10 minutes;Hold at 10C until next step</li></ol> | |||
Continued [[Daniel:Notebook/ComboLock/2017-1-31|tomorrow]] | |||
[[Category:ComboLock]] [[Category:20170127]] [[Category:20170123]] |
Latest revision as of 23:59, 30 January 2017
RCA Test (Started Monday)[edit]
Library Prep (Started Jan 26)[edit]
- Ethanol Precipitation
- Centrifuge for 30 minutes at 12500 rpm at 4C
- Remove supernatant and add 750 uL chilled 70% EtOH
- Centrifuge for 15 minutes at 12500 rpm at 4C
- Remove supernatant; dry in vacuum centrifuge for 15 minutes
- Resuspend in 20 uL H2O and consolidate samples
- Measure with Qbit
- Consolidate with samples from Thursday 1-26
DEJ20160126-CLv3-RCA02
Final Sample Matrix
Sample | Sample Amt | Pre/Post RCA | AmpR Index |
Sample 0 (AB) | 1 pmol | Pre | 21 |
Sample 0X (AB) | 1 pmol | Post | 25 |
Sample 2X (AB) | 10 fmol | Post | 26 |
Sample 4X (AB) | 100 amol | Post | 27 |
Sample 6C (AB) | 1 amol | Epi | 22 |
Sample 6D (AB) | 1 amol | Epi 1:10 | 23 |
RCA Timing Test (Started Friday)[edit]
Because of the results from Saturday, I think I want to try Epicentre again for as long as possible. I'll do 12 hours with (as usual) one negative control to save up on epicentre enzyme.
- Rolling Circle Amplification
- Prepare master mix according to table below
- Add 14 uL master mix to each tube
- Add sample and to new reaction tubes according to following table
- Incubate at 30C for 12 hours
- Incubate at 65C for 10 minutes;Hold at 10C until next step
Reagent | uL Added | Master Mix (4.2X) |
Template | 4 | NA |
RCA Primer (10 uM) | 2.5 | 10.5 |
dNTP (1 mM) | 2 | 8.4 |
10X Buffer | 2 | 8.4 |
Phi29 | 1 | NA |
nfH2O | 8.5 | 35.7 |
Total | 20 | 63 |
Continued tomorrow