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>Djacobse (Created page with "=Mirror Probe Test (Started Yesterday)= Back to Calendar ==Protocol== <ol start="5"> <li>Exonuclease ...") |
>Djacobse |
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(11 intermediate revisions by the same user not shown) | |||
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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>151.2 uL nfH2O</li> | ||
<li> | <li>180 uL SYBR Fast</li> | ||
<li>7. | <li>7.2 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 187: | Line 187: | ||
<li>TBE Gel</li> | <li>TBE Gel</li> | ||
<ol type="A"> | <ol type="A"> | ||
<li>Mix | <li>Mix 80 uL TBE, 20 uL 6x loading dye</li> | ||
<li>Aliquot 10 uL per sample/ladder lane onto parafilm</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>Add 2 uL of sample or ladder to correct drop</li> | ||
Line 199: | Line 199: | ||
<gallery perrow=2 heights=300px widths=300px> | <gallery perrow=2 heights=300px widths=300px> | ||
File:|qPCR Curve | File:20161214-qPCR-MirrorCProbe.png|qPCR Curve | ||
File:|Gel image | File:2016-12-14-MirrorCProbes.png|Gel image | ||
</gallery> | </gallery> | ||
So, nothing on this looks good. The PCR was more promising, with the sample lanes and (less encouragingly) N C Probe lanes showing a slightly higher CT than the No Template which was better than the NTC. | |||
I'm going to try looking at the beads effect more closely now. I'll do PCR off the supernatant and beads fractions, as well as RCA on the beads fraction to test. | |||
==Beads vs Supernatant Fractions== | |||
I also might have neglected the dNTPs for RCA earlier. Just to make sure, I'm also doing a supernatant fraction RCA amplification. Protocol is the same for both. | |||
<ol start="6"> | |||
<li>Rolling Circle Amplification</li> | |||
<ol type="A"> | |||
<li>Prepare 7X Master mix according to table below</li> | |||
<li>Add 15 uL master mix to each tube</li> | |||
<li>Add sample to new reaction tubes according to following 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="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> | |||
</ol> | |||
Continued [[Daniel:Notebook/ComboLock/2016-12-15|tomorrow]] | |||
==Pre-RCA Beads vs Supernatant== | |||
<ol start="7"> | |||
<li>qPCR</li> | |||
<ol type="A"> | |||
<li>Make master mix according to following recipe</li> | |||
<ol type="a"> | |||
<li>298.2 uL nfH2O</li> | |||
<li>355 uL SYBR Fast</li> | |||
<li>14.2 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 in the following section</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="90" height="45" | Sample | |||
| width="120" | Condition | |||
| width="100" | Fraction | |||
| width="85" | Lane | |||
| width="85" | AmpR Index | |||
| width="85" | Sample Vol (uL) | |||
| width="105" | 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" | Supernatant | |||
| 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" | Supernatant | |||
| 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" | Supernatant | |||
| 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" | Supernatant | |||
| 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 C Probe | |||
| align="center" | Supernatant | |||
| 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 C Probe | |||
| align="center" | Supernatant | |||
| 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 1A | |||
| align="center" | Sample | |||
| align="center" | Beads | |||
| align="center" | H1 | |||
| 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:#DA9694;font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 1B | |||
| align="center" | Sample | |||
| align="center" | Beads | |||
| align="center" | H2 | |||
| 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 2A | |||
| align="center" | No Template | |||
| align="center" | Beads | |||
| align="center" | H3 | |||
| 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:#DA9694;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | Sample 2B | |||
| align="center" valign="bottom" | No Template | |||
| align="center" | Beads | |||
| align="center" | H4 | |||
| 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 3A | |||
| align="center" | No C Probe | |||
| align="center" | Beads | |||
| align="center" | H5 | |||
| 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:#DA9694;font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 3B | |||
| align="center" | No C Probe | |||
| align="center" | Beads | |||
| align="center" | H6 | |||
| 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" | |||
| height="30" valign="bottom" | NTC | |||
| align="center" valign="bottom" | qPCR Primers Only | |||
| align="center" valign="bottom" | NA | |||
| align="center" | A7 | |||
| 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" | 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 80 uL TBE, 20 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=3 heights=300px widths=300px> | |||
File:20161214-qPCR-MirrorCProbe-PreRCA.png|qPCR Image | |||
File:2016-12-14-MirrorCProbes-PreRCA-Supernatant.png|Gel image-Supernatant Pre-RCA | |||
File:2016-12-14-MirrorCProbes-PreRCA-Beads.png|Gel image-Pre-RCA Beads | |||
</gallery> | |||
[[Category:ComboLock]] [[Category:20161213]] |
Latest revision as of 00:57, 15 December 2016
Mirror Probe Test (Started Yesterday)[edit]
Protocol[edit]
- 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
- 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 in the following section
- 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 80 uL TBE, 20 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 | 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 | 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 | A1 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1B | Sample | A2 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2A | No Template | A3 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2B | No Template | A4 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3A | No C Probe | A5 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3B | No C Probe | A6 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
NTC | qPCR Primers Only | A7 | 23 | 2 | 25 | 1 | 1 | 23 | 50 |
Results[edit]
- 20161214-qPCR-MirrorCProbe.png
qPCR Curve
- 2016-12-14-MirrorCProbes.png
Gel image
So, nothing on this looks good. The PCR was more promising, with the sample lanes and (less encouragingly) N C Probe lanes showing a slightly higher CT than the No Template which was better than the NTC.
I'm going to try looking at the beads effect more closely now. I'll do PCR off the supernatant and beads fractions, as well as RCA on the beads fraction to test.
Beads vs Supernatant Fractions[edit]
I also might have neglected the dNTPs for RCA earlier. Just to make sure, I'm also doing a supernatant fraction RCA amplification. Protocol is the same for both.
- 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
- Incubate at 37C for 3 hours
- Incubate at 65C for 10 minutes;Hold at 10C until next step
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 |
Continued tomorrow
Pre-RCA Beads vs Supernatant[edit]
- qPCR
- Make master mix according to following recipe
- 298.2 uL nfH2O
- 355 uL SYBR Fast
- 14.2 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 in the following section
- 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 80 uL TBE, 20 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
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 | H1 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1B | Sample | Beads | H2 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2A | No Template | Beads | H3 | 24 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2B | No Template | Beads | H4 | 24 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3A | No C Probe | Beads | H5 | 25 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3B | No C Probe | Beads | H6 | 25 | 2 | 25 | 1 | 1 | 21 | 50 |
NTC | qPCR Primers Only | NA | A7 | 26 | 2 | 25 | 1 | 1 | 23 | 50 |
Results[edit]
- 20161214-qPCR-MirrorCProbe-PreRCA.png
qPCR Image
- 2016-12-14-MirrorCProbes-PreRCA-Supernatant.png
Gel image-Supernatant Pre-RCA
- 2016-12-14-MirrorCProbes-PreRCA-Beads.png
Gel image-Pre-RCA Beads