Daniel:Notebook/ComboLock/2017-1-26: Difference between revisions
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<ol type="a"> | <ol type="a"> | ||
<li>109.2 uL nfH2o</li> | <li>109.2 uL nfH2o</li> | ||
<li>5.2 uL AmpF | <li>5.2 uL AmpF</li> | ||
<li>130 uL 2X Kapa SYBR Fast Master Mix</li> | <li>130 uL 2X Kapa SYBR Fast Master Mix</li> | ||
<li>Add 47 uL master mix to each well</li> | <li>Add 47 uL master mix to each well</li> | ||
Line 114: | Line 114: | ||
<li>Open gel and stain with 3 uL SYBR Gold for 3 minutes</li> | <li>Open gel and stain with 3 uL SYBR Gold for 3 minutes</li> | ||
<li>Rinse gel and image in gel doc</li> | <li>Rinse gel and image in gel doc</li> | ||
</ol></ol> | |||
==Library Prep== | |||
Those bands are interesting to me. Time to sequence. | |||
'''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 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 | |||
|} | |||
<ol> | |||
<li>Size Select Gel</li> | |||
<ol type="A"> | |||
<li>Mix 20 uL sample from each tech rep lane (A and B) and 8 uL 6x loading dye into separate tubes for each sample (Samples 1A and 1B)</li> | |||
<li>Mix 4 uL ladder, 4 uL 6x dye, and 16 uL TBE in ladder tube</li> | |||
<li>Aliquot 48/24 uL per sample/ladder lane into each well</li> | |||
<li>Run gel for 23 minutes at 230V</li> | |||
<li>Open gel and stain with 3 uL SYBR Gold for 3 minutes</li> | |||
<li>Rinse gel and image in gel doc</li> | |||
<li>Cut out appropriate bands with scalpel; put in 0.5 mL tube with hole in the bottom inside a 1.5 mL tube</li> | |||
<li>Image in gel doc</li> | |||
<gallery perrow=2 heights=300px widths=300px> | |||
File:2017-01-26-RCATest-Epicentre-SizeSelect.png|Before Image | |||
File:2017-01-26-RCATest-Epicentre-SizeSelect-After.png|After Image | |||
</gallery> | |||
<li>Centrifuge at 12000 rpm for 1.5 minutes</li> | |||
<li>Add 500 uL TE to each sample</li> | |||
<li>Incubate 4 hours at 37C with vigorous shaking;</li> | |||
<li>Centrifuge tubes at 12000 rpm for 1.5 minutes to bring gel to bottom</li> | |||
<li>Extract supernatant and add to nanosep column</li> | |||
<li>Centrifuge at 12000 rpm for 1.5 minutes; collect supernatant</li></ol> | |||
<li>Ethanol Precipitation</li> | |||
<ol type="A"> | |||
<li>Add 1250 uL 100% EtOH, 50 uL 3M NaOAc, and 1.5 uL glycoblue to each sample</li> | |||
<li>Incubate at -80C overnight; continued [[Daniel:Notebook/ComboLock/2017-1-27|tomorrow]]</li> | |||
</ol></ol> | </ol></ol> | ||
Line 122: | Line 176: | ||
File:2017-01-26-RCATest-Epicentre.png|Gel image | File:2017-01-26-RCATest-Epicentre.png|Gel image | ||
</gallery> | </gallery> | ||
==Library Prep (Started [[Daniel:Notebook/ComboLock/2017-1-25|Yesterday]])== | |||
<ol start="2"> | |||
<li>Ethanol Precipitation</li> | |||
<ol type="A" start="3"> | |||
<li>Centrifuge for 30 minutes at 12500 rpm at 4C</li> | |||
<li>Remove supernatant and add 750 uL chilled 70% EtOH</li> | |||
<li>Centrifuge for 15 minutes at 12500 rpm at 4C</li> | |||
<li>Remove supernatant; dry in vacuum centrifuge for 15 minutes</li> | |||
<li>Resuspend in 20 uL H2O and consolidate samples</li> | |||
<li>Measure with Qbit</li> | |||
</ol></ol> | |||
[[Category:ComboLock]] [[Category:20170123]] |
Latest revision as of 23:22, 30 January 2017
RCA Test (Started Monday)[edit]
Epicentre Test (Started Yesterday)[edit]
Sample Matrix
- qPCR
- Make 5.2X qPCR master mix according to following recipe
- 109.2 uL nfH2o
- 5.2 uL AmpF
- 130 uL 2X Kapa SYBR Fast Master Mix
- 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 64 uL TBE, 16 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 23 minutes at 230V
- Open gel and stain with 3 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc
- Size Select Gel
- Mix 20 uL sample from each tech rep lane (A and B) and 8 uL 6x loading dye into separate tubes for each sample (Samples 1A and 1B)
- Mix 4 uL ladder, 4 uL 6x dye, and 16 uL TBE in ladder tube
- Aliquot 48/24 uL per sample/ladder lane into each well
- Run gel for 23 minutes at 230V
- Open gel and stain with 3 uL SYBR Gold for 3 minutes
- Rinse gel and image in gel doc
- Cut out appropriate bands with scalpel; put in 0.5 mL tube with hole in the bottom inside a 1.5 mL tube
- Image in gel doc
- 2017-01-26-RCATest-Epicentre-SizeSelect.png
Before Image
- 2017-01-26-RCATest-Epicentre-SizeSelect-After.png
After Image
- Centrifuge at 12000 rpm for 1.5 minutes
- Add 500 uL TE to each sample
- Incubate 4 hours at 37C with vigorous shaking;
- Centrifuge tubes at 12000 rpm for 1.5 minutes to bring gel to bottom
- Extract supernatant and add to nanosep column
- Centrifuge at 12000 rpm for 1.5 minutes; collect supernatant
- Ethanol Precipitation
- Add 1250 uL 100% EtOH, 50 uL 3M NaOAc, and 1.5 uL glycoblue to each sample
- Incubate at -80C overnight; continued tomorrow
- 20170126-qPCR-RCATest-Epicentre.png
qPCR curve
- 2017-01-26-RCATest-Epicentre.png
Gel image
- 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
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 6AC | Epicentre | A1 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 6BC | Epicentre | A2 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 6AD | Epicentre 1:10 | A3 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 6BD | Epicentre 1:10 | A4 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
NTC | A5 | 24 | 2 | 25 | 1 | 1 | 21 | 50 |
Library Prep[edit]
Those bands are interesting to me. Time to sequence.
Sample Matrix
Sample | Sample Amt | Pre/Post RCA | AmpR Index |
Sample 6C (AB) | 1 amol | Epi | 22 |
Sample 6D (AB) | 1 amol | Epi 1:10 | 23 |