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>Djacobse (Created page with "=Extended Latch Test (Started [[Daniel:Notebook/ComboLock/2016-11-29])= Back to Calendar ===Sample Matrix=== {| class="wikitable" <hiddentext>...") |
>Djacobse |
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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]] | ||
Line 20: | Line 20: | ||
|- 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 | ||
|} | |} | ||
Line 43: | Line 43: | ||
{| 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:#C4D79B;font-size:12pt;font-weight:bold" align="center" | |- style="background-color:#C4D79B;font-size:12pt;font-weight:bold" align="center" | ||
| width=" | | width="160" height="30" | Reagent | ||
| width="94" | Stock Concentration | | width="94" | Stock Concentration | ||
| width="77" | Final Conc/Amt | | width="77" | Final Conc/Amt | ||
Line 57: | Line 57: | ||
|- style="background-color:#BFBFBF;font-size:12pt" | |- style="background-color:#BFBFBF;font-size:12pt" | ||
| height="15" valign="bottom" | RCA Primer | | height="15" valign="bottom" | RCA Primer (RCA-CL-LinkerLRC-v01) | ||
| align="center" valign="bottom" | 10 uM | | align="center" valign="bottom" | 10 uM | ||
| align="center" valign="bottom" | 2 uM | | align="center" valign="bottom" | 2 uM | ||
Line 104: | Line 104: | ||
<li>Incubate at 37C for 3 hours</li> | <li>Incubate at 37C for 3 hours</li> | ||
<li>Heat kill by incubating at 65C for 10 minutes</li></ol> | <li>Heat kill by incubating at 65C for 10 minutes</li></ol> | ||
<li>qPCR</li> | |||
<ol type="A"> | |||
<li>Make master mix according to following recipe</li> | |||
<ol type="a"> | |||
<li>359.1 uL nfH2O</li> | |||
<li>427.5 uL SYBR Fast</li> | |||
<li>17.1 uL 10 uM AmpF</li> | |||
</ol> | </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> | |||
|- style="background-color:#92CDDC;font-size:12pt;font-weight:bold" align="center" | |||
| width="85" height="45" | Sample | |||
| width="120" | Condition | |||
| width="85" | RCA | |||
| width="85" | Lane | |||
| width="85" | AmpR Index | |||
| width="85" | Sample Vol (uL) | |||
| width="85" | 2X Kapa SYBR qPCR MM | |||
| width="85" | 10 uM Forward Primer | |||
| width="85" | 10 uM Reverse Primer | |||
| width="85" | H2O | |||
| width="85" | Total Volume (uL) | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 1A | |||
| align="center" | Sample | |||
| align="center" | Y | |||
| align="center" | A1 | |||
| align="center" align="center" | 20 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 1B | |||
| align="center" | Sample | |||
| align="center" | Y | |||
| align="center" | A2 | |||
| align="center" align="center" | 20 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 2A | |||
| align="center" | No Template | |||
| align="center" | Y | |||
| align="center" | A3 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Sample 2B | |||
| align="center" valign="bottom" | No Template | |||
| align="center" valign="bottom" | Y | |||
| align="center" | A4 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 3A | |||
| align="center" | No Phosphate | |||
| align="center" | Y | |||
| align="center" | A5 | |||
| align="center" align="center" | 22 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 3B | |||
| align="center" | No Phosphate | |||
| align="center" | Y | |||
| align="center" | A6 | |||
| align="center" align="center" | 22 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 4A | |||
| align="center" | No C Probe | |||
| align="center" | Y | |||
| align="center" | A7 | |||
| align="center" align="center" | 23 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 4B | |||
| align="center" | No C Probe | |||
| align="center" | Y | |||
| align="center" | A8 | |||
| align="center" align="center" | 23 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 1A | |||
| align="center" | Sample | |||
| align="center" | N | |||
| align="center" | D1 | |||
| align="center" align="center" | 24 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 1B | |||
| align="center" | Sample | |||
| align="center" | N | |||
| align="center" | D2 | |||
| align="center" align="center" | 24 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 2A | |||
| align="center" | No Template | |||
| align="center" | N | |||
| align="center" | D3 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Sample 2B | |||
| align="center" valign="bottom" | No Template | |||
| align="center" valign="bottom" | N | |||
| align="center" | D4 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 3A | |||
| align="center" | No Phosphate | |||
| align="center" | N | |||
| align="center" | D5 | |||
| align="center" align="center" | 26 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 3B | |||
| align="center" | No Phosphate | |||
| align="center" | N | |||
| align="center" | D6 | |||
| align="center" align="center" | 26 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 4A | |||
| align="center" | No C Probe | |||
| align="center" | N | |||
| align="center" | D7 | |||
| align="center" align="center" | 27 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="background-color:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 4B | |||
| align="center" | No C Probe | |||
| align="center" | N | |||
| align="center" | D8 | |||
| align="center" align="center" | 27 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 21 | |||
| align="center" align="center" | 50 | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="30" valign="bottom" | NTC | |||
| align="center" valign="bottom" | qPCR Primers Only | |||
| align="center" valign="bottom" | NA | |||
| align="center" | H1 | |||
| align="center" align="center" | 28 | |||
| align="center" align="center" | 2 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 1 | |||
| align="center" align="center" | 23 | |||
| align="center" align="center" | 50 | |||
|} | |||
<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 x24</li> | |||
<li>72C 2 min</li> | |||
<li>16C hold</li> | |||
</ol></ol> | |||
<li>TBE Gel</li> | |||
<ol type="A"> | |||
<li>Mix 200 uL TBE, 50 uL 6x loading dye</li> | |||
<li>Aliquot 10 uL per sample/ladder lane onto parafilm</li> | |||
<li>Add 2 uL of sample or ladder to correct drop</li> | |||
<li>Load 10 uL in to well</li> | |||
<li>Run gel for 22 minutes at 250V</li> | |||
<li>Open gel and stain with 3 uL SYBR Gold for 3 minutes</li> | |||
<li>Rinse gel and image in gel doc</li> | |||
</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.