Daniel:Notebook/ComboLock/2016-11-30: Difference between revisions
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=Extended Latch Test (Started [[Daniel:Notebook/ComboLock/2016-11-29]])= | =Extended Latch Test (Started [[Daniel:Notebook/ComboLock/2016-11-29|Nov 29]])= | ||
[[Daniel:Notebook/ComboLock|Back to Calendar]] | [[Daniel:Notebook/ComboLock|Back to Calendar]] | ||
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|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | Sample 3 (AB) | | height="15" valign="bottom" | Sample 3 (AB) | ||
| align="center" valign="bottom" | No | | align="center" valign="bottom" | No Phosphate Ligation | ||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| height="15" valign="bottom" | Sample 4 (AB) | | height="15" valign="bottom" | Sample 4 (AB) | ||
| align="center" valign="bottom" | No | | align="center" valign="bottom" | No C Probe | ||
|} | |} | ||
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<li>Make master mix according to following recipe</li> | <li>Make master mix according to following recipe</li> | ||
<ol type="a"> | <ol type="a"> | ||
<li> | <li>359.1 uL nfH2O</li> | ||
<li> | <li>427.5 uL SYBR Fast</li> | ||
<li> | <li>17.1 uL 10 uM AmpF</li> | ||
</ol> | </ol> | ||
<li>Add 47 uL master mix to each well</li> | <li>Add 47 uL master mix to each well</li> | ||
Line 117: | Line 117: | ||
{| 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="85" height=" | | width="85" height="45" | Sample | ||
| width=" | | width="120" | Condition | ||
| width=" | | width="85" | RCA | ||
| width="85" | Lane | | width="85" | Lane | ||
| width="85" | AmpR Index | | width="85" | AmpR Index | ||
| width="85" | Sample Vol (uL) | | width="85" | Sample Vol (uL) | ||
| width=" | | width="85" | 2X Kapa SYBR qPCR MM | ||
| width=" | | width="85" | 10 uM Forward Primer | ||
| width=" | | width="85" | 10 uM Reverse Primer | ||
| width="85" | H2O | | width="85" | H2O | ||
| width=" | | width="85" | Total Volume (uL) | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
Line 237: | Line 237: | ||
| align="center" | Sample | | align="center" | Sample | ||
| align="center" | N | | align="center" | N | ||
| align="center" | | | align="center" | D1 | ||
| align="center" align="center" | 24 | | align="center" align="center" | 24 | ||
| align="center" align="center" | 2 | | align="center" align="center" | 2 | ||
Line 250: | Line 250: | ||
| align="center" | Sample | | align="center" | Sample | ||
| align="center" | N | | align="center" | N | ||
| align="center" | | | align="center" | D2 | ||
| align="center" align="center" | 24 | | align="center" align="center" | 24 | ||
| align="center" align="center" | 2 | | align="center" align="center" | 2 | ||
Line 263: | Line 263: | ||
| align="center" | No Template | | align="center" | No Template | ||
| align="center" | N | | align="center" | N | ||
| align="center" | | | align="center" | D3 | ||
| align="center" align="center" | 25 | | align="center" align="center" | 25 | ||
| align="center" align="center" | 2 | | align="center" align="center" | 2 | ||
Line 276: | Line 276: | ||
| align="center" valign="bottom" | No Template | | align="center" valign="bottom" | No Template | ||
| align="center" valign="bottom" | N | | align="center" valign="bottom" | N | ||
| align="center" | | | align="center" | D4 | ||
| align="center" align="center" | 25 | | align="center" align="center" | 25 | ||
| align="center" align="center" | 2 | | align="center" align="center" | 2 | ||
Line 289: | Line 289: | ||
| align="center" | No Phosphate | | align="center" | No Phosphate | ||
| align="center" | N | | align="center" | N | ||
| align="center" | | | align="center" | D5 | ||
| align="center" align="center" | 26 | | align="center" align="center" | 26 | ||
| align="center" align="center" | 2 | | align="center" align="center" | 2 | ||
Line 302: | Line 302: | ||
| align="center" | No Phosphate | | align="center" | No Phosphate | ||
| align="center" | N | | align="center" | N | ||
| align="center" | | | align="center" | D6 | ||
| align="center" align="center" | 26 | | align="center" align="center" | 26 | ||
| align="center" align="center" | 2 | | align="center" align="center" | 2 | ||
Line 315: | Line 315: | ||
| align="center" | No C Probe | | align="center" | No C Probe | ||
| align="center" | N | | align="center" | N | ||
| align="center" | | | align="center" | D7 | ||
| align="center" align="center" | 27 | | align="center" align="center" | 27 | ||
| align="center" align="center" | 2 | | align="center" align="center" | 2 | ||
Line 328: | Line 328: | ||
| align="center" | No C Probe | | align="center" | No C Probe | ||
| align="center" | N | | align="center" | N | ||
| align="center" | | | align="center" | D8 | ||
| align="center" align="center" | 27 | | align="center" align="center" | 27 | ||
| align="center" align="center" | 2 | | align="center" align="center" | 2 | ||
Line 338: | Line 338: | ||
|- style="font-size:12pt" | |- style="font-size:12pt" | ||
|style="font-weight:bold" height=" | |style="font-weight:bold" height="30" valign="bottom" | NTC | ||
| align="center" valign="bottom" | qPCR Primers Only | | align="center" valign="bottom" | qPCR Primers Only | ||
| align="center" valign="bottom" | NA | | align="center" valign="bottom" | NA | ||
Line 373: | Line 373: | ||
<li>Rinse gel and image in gel doc</li> | <li>Rinse gel and image in gel doc</li> | ||
</ol></ol> | </ol></ol> | ||
===Results=== | |||
<gallery perrow=2 heights=300px widths=300px> | |||
File:20161130-qPCR-ExtLatch.png|qPCR image | |||
File:2016-11-30-ExtLatch-RCA.png|Gel image (RCA +) | |||
File:2016-11-30-ExtLatch-NoRCA.png|Gel image (Before RCA) | |||
</gallery> | |||
You can see that the pre and post-RCA reactions amplify at the same rate and are even similar in their gel smears. [[Daniel:Notebook/ComboLock/2016-12-1|Tomorrow]] I will do some QC testing and try to find where the problems are. | |||
[[Category:ComboLock]] [[Category:20161129]] |
Latest revision as of 23:14, 1 December 2016
Extended Latch Test (Started Nov 29)[edit]
Sample Matrix[edit]
Sample | Condition |
Sample 1 (AB) | Normal |
Sample 2 (AB) | No Template |
Sample 3 (AB) | No Phosphate Ligation |
Sample 4 (AB) | No C Probe |
Protocol[edit]
- Exonuclease Digestion
- Combine 15 uL of Exonuclease I (20U/uL) and 3 uL Exonuclease III (100U/uL)
- Add 2 uL of mix to each reaction
- Incubate for 30 minutes to 2 hours at 37C
- Heat kill by incubating for 5 minutes at 94C
- Rolling Circle Amplification
- Prepare 9X master mix according to table below
- Add 15 uL master mix to each tube
- Add 5 uL sample to correct reaction tubes
- Incubate at 37C for 3 hours
- Heat kill by incubating at 65C for 10 minutes
- qPCR
- Make master mix according to following recipe
- 359.1 uL nfH2O
- 427.5 uL SYBR Fast
- 17.1 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
- qPCR Cycles
- 95C 3 min
- 95C 3 sec
- 55C 30 sec
- 72C 20 sec
- plate read
- goto b x24
- 72C 2 min
- 16C hold
- TBE Gel
- Mix 200 uL TBE, 50 uL 6x loading dye
- Aliquot 10 uL per sample/ladder lane onto parafilm
- Add 2 uL of sample or ladder to correct drop
- Load 10 uL in to well
- Run gel for 22 minutes at 250V
- Open gel and stain with 3 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc
Reagent | Stock Concentration | Final Conc/Amt | uL Added in 20 uL Rxn | Master Mix (9X) |
Template | NA | NA | 5 | 45 |
RCA Primer (RCA-CL-LinkerLRC-v01) | 10 uM | 2 uM | 4 | 36 |
dNTP | 1 mM | 50 uM | 1 | 9 |
10X Buffer | 10X | 1X | 2 | 18 |
Phi29 (NEB) | 10 U/uL | 10 U | 1 | 9 |
nfH2O | NA | NA | 7 | 63 |
Total | 20 | 180 |
Sample | Condition | RCA | 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 | Y | A1 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1B | Sample | Y | A2 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2A | No Template | Y | A3 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2B | No Template | Y | A4 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3A | No Phosphate | Y | A5 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3B | No Phosphate | Y | A6 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4A | No C Probe | Y | A7 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4B | No C Probe | Y | A8 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1A | Sample | N | D1 | 24 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1B | Sample | N | D2 | 24 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2A | No Template | N | D3 | 25 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2B | No Template | N | D4 | 25 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3A | No Phosphate | N | D5 | 26 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3B | No Phosphate | N | D6 | 26 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4A | No C Probe | N | D7 | 27 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4B | No C Probe | N | D8 | 27 | 2 | 25 | 1 | 1 | 21 | 50 |
NTC | qPCR Primers Only | NA | H1 | 28 | 2 | 25 | 1 | 1 | 23 | 50 |
Results[edit]
- 20161130-qPCR-ExtLatch.png
qPCR image
- 2016-11-30-ExtLatch-RCA.png
Gel image (RCA +)
- 2016-11-30-ExtLatch-NoRCA.png
Gel image (Before RCA)
You can see that the pre and post-RCA reactions amplify at the same rate and are even similar in their gel smears. Tomorrow I will do some QC testing and try to find where the problems are.