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>Djacobse (Created page with "=BSA Beads Test (Started Yesterday)= Back to Calendar ==Protocol== <li>Rolling Circle Amplification</...") |
>Djacobse |
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(13 intermediate revisions by the same user not shown) | |||
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==Protocol== | ==Protocol== | ||
<ol start="5"> | |||
<li>Rolling Circle Amplification</li> | <li>Rolling Circle Amplification</li> | ||
<ol type="A"> | <ol type="A"> | ||
Line 56: | Line 57: | ||
<li>Incubate at 65C for 10 minutes;Hold at 10C until next step</li></ol> | <li>Incubate at 65C for 10 minutes;Hold at 10C until next step</li></ol> | ||
<li>qPCR</li> | <li>qPCR</li> | ||
<ol type="A"> | |||
<li>Make master mix according to following recipe</li> | |||
<ol type="a"> | |||
<li>422.1 uL nfH2O</li> | |||
<li>502.5 uL SYBR Fast</li> | |||
<li>20.1 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 in the following section</li> | |||
<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 widths=300px heights=300px> | |||
File:20161116-qPCR-BSABeads-ChrisPrimer.png|qPCR curve; Chris' RCA primer | |||
File:20161116-qPCR-BSABeads-DanPrimer.png|qPCR curve; Dan's RCA primer | |||
File:2016-11-16-RCA-BSA-ChrisPrimer.png|Gel image 1; Chris' RCA Primer | |||
File:2016-11-16-RCA-BSA-DanPrimer.png|Gel image 2; Dan's RCA Primer | |||
</gallery> | |||
===qPCR Sample Table=== | |||
{| 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="120" height="45" | Sample | |||
| width="82" | RCA-Primer | |||
| width="130" | Condition | |||
| width="65" | Lane | |||
| width="65" | 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="65" | H2O | |||
| width="90" | Total Volume (uL) | |||
|- style="font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 1AC | |||
| align="center" | Chris | |||
|style="font-weight:bold" align="center" | 2 ug BSA-biotin | |||
| 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 1BC | |||
| align="center" | Chris | |||
|style="font-weight:bold" align="center" | 2 ug BSA-biotin | |||
| 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 2AC | |||
| align="center" | Chris | |||
|style="font-weight:bold" align="center" | No BSA-biotin | |||
| 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 2BC | |||
| align="center" | Chris | |||
|style="font-weight:bold" align="center" valign="bottom" | No BSA-biotin | |||
| 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 3AC | |||
| align="center" | Chris | |||
|style="font-weight:bold" align="center" | No Antibody | |||
| 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 3BC | |||
| align="center" | Chris | |||
|style="font-weight:bold" align="center" | No Antibody | |||
| 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 4AC | |||
| align="center" | Chris | |||
|style="font-weight:bold" align="center" | 20 ng BSA-biotin | |||
| 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" valign="bottom" | Sample 4BC | |||
| align="center" | Chris | |||
|style="font-weight:bold" align="center" valign="bottom" | 20 ng BSA-biotin | |||
| 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 5AC | |||
| align="center" | Chris | |||
|style="font-weight:bold" align="center" | 200 pg BSA-biotin | |||
| 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 5BC | |||
| align="center" | Chris | |||
|style="font-weight:bold" align="center" | 200 pg BSA-biotin | |||
| 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 1AD | |||
| align="center" | Dan | |||
|style="font-weight:bold" align="center" | 2 ug BSA-biotin | |||
| align="center" | D3 | |||
| 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" valign="bottom" | Sample 1BD | |||
| align="center" valign="bottom" | Dan | |||
|style="font-weight:bold" align="center" valign="bottom" | 2 ug BSA-biotin | |||
| align="center" | D4 | |||
| 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 2AD | |||
| align="center" | Dan | |||
|style="font-weight:bold" align="center" | No BSA-biotin | |||
| align="center" | D5 | |||
| 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" | Sample 2BD | |||
| align="center" valign="bottom" | Dan | |||
|style="font-weight:bold" align="center" | No BSA-biotin | |||
| align="center" | D6 | |||
| 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 3AD | |||
| align="center" | Dan | |||
|style="font-weight:bold" align="center" | No Antibody | |||
| align="center" | D7 | |||
| 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" valign="bottom" | Sample 3BD | |||
| align="center" valign="bottom" | Dan | |||
|style="font-weight:bold" align="center" valign="bottom" | No Antibody | |||
| align="center" | D8 | |||
| 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 4AD | |||
| align="center" | Dan | |||
|style="font-weight:bold" align="center" | 20 ng BSA-biotin | |||
| 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:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" | Sample 4BD | |||
| align="center" valign="bottom" | Dan | |||
|style="font-weight:bold" align="center" | 20 ng BSA-biotin | |||
| 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 5AD | |||
| align="center" | Dan | |||
|style="font-weight:bold" align="center" | 200 pg BSA-biotin | |||
| 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:#BFBFBF;font-size:12pt" | |||
|style="font-weight:bold" height="15" valign="bottom" | NTC | |||
| align="center" valign="bottom" | | |||
|style="font-weight:bold" align="center" align="center" valign="bottom" | | |||
| align="center" | H4 | |||
| align="center" align="center" | 25 | |||
| align="center" align="center" | 0 | |||
| 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 | |||
|} | |||
=Library Prep (Started [[Daniel:Notebook/ComboLock/2016-11-15|Yesterday]]; Exp. Start [[Daniel:Notebook/ComboLock/2016-11-7|11/7/16]])= | |||
<ol start="2"> | |||
<li>Ethanol Precipitation</li> | |||
<ol type="A" start="2"> | |||
<li>Centrifuge for 25 minutes at 12000 rpm at 4C</li> | |||
<li>Remove supernatant and add 750 uL 70% EtOH</li> | |||
<li>Centrifuge for 12 minutes at 12000 rpm at 4C</li> | |||
<li>Remove supernatant and spin in vacuum centrifuge for 10 minutes</li> | |||
<li>Add 30 (20 for NTC) uL nfH2O to resuspend; pool samples A and B by reusing the water</li> | |||
</ol></ol> | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#C4BD97;font-size:12pt;font-weight:bold" align="center" | |||
| width="160" height="34" | Sample | |||
| width="85" | ng/uL | |||
| width="85" | ug in 30 uL | |||
|- style="font-size:12pt" | |||
| height="30" valign="bottom" | Cells RCA Product (2AB,2BB) | |||
| align="center" align="center" | 47.8 | |||
| align="center" align="center" | 1.4 | |||
|} | |||
'''DEJ-CL-CellC01-20161116-Ind25''' | |||
[[Category:ComboLock]] [[Category:20161107]] [[Category:20161115]] |
Latest revision as of 21:13, 21 November 2016
BSA Beads Test (Started Yesterday)[edit]
Protocol[edit]
- Rolling Circle Amplification
- Prepare 10.2X Master mix according to table below
- 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
- 422.1 uL nfH2O
- 502.5 uL SYBR Fast
- 20.1 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 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 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 | uL Added | Master Mix (10.2X) |
Template | 5 | NA |
RCA Primer (100 uM) | 2.5 | 25.5 |
dNTP (1 mM) | 0.8 | 8.16 |
10X Buffer | 2 | 20.4 |
Phi29 | 1 | 10.2 |
nfH2O | 8.7 | 88.74 |
Total | 20 | 153 |
Results[edit]
- 20161116-qPCR-BSABeads-ChrisPrimer.png
qPCR curve; Chris' RCA primer
- 20161116-qPCR-BSABeads-DanPrimer.png
qPCR curve; Dan's RCA primer
- 2016-11-16-RCA-BSA-ChrisPrimer.png
Gel image 1; Chris' RCA Primer
- 2016-11-16-RCA-BSA-DanPrimer.png
Gel image 2; Dan's RCA Primer
qPCR Sample Table[edit]
Sample | RCA-Primer | 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 1AC | Chris | 2 ug BSA-biotin | A1 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1BC | Chris | 2 ug BSA-biotin | A2 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2AC | Chris | No BSA-biotin | A3 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2BC | Chris | No BSA-biotin | A4 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3AC | Chris | No Antibody | A5 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3BC | Chris | No Antibody | A6 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4AC | Chris | 20 ng BSA-biotin | A7 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4BC | Chris | 20 ng BSA-biotin | A8 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 5AC | Chris | 200 pg BSA-biotin | D1 | 24 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 5BC | Chris | 200 pg BSA-biotin | D2 | 24 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1AD | Dan | 2 ug BSA-biotin | D3 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 1BD | Dan | 2 ug BSA-biotin | D4 | 20 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2AD | Dan | No BSA-biotin | D5 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2BD | Dan | No BSA-biotin | D6 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3AD | Dan | No Antibody | D7 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 3BD | Dan | No Antibody | D8 | 22 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4AD | Dan | 20 ng BSA-biotin | H1 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 4BD | Dan | 20 ng BSA-biotin | H2 | 23 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 5AD | Dan | 200 pg BSA-biotin | H3 | 24 | 2 | 25 | 1 | 1 | 21 | 50 |
NTC | H4 | 25 | 0 | 25 | 1 | 1 | 23 | 50 |
Library Prep (Started Yesterday; Exp. Start 11/7/16)[edit]
- Ethanol Precipitation
- Centrifuge for 25 minutes at 12000 rpm at 4C
- Remove supernatant and add 750 uL 70% EtOH
- Centrifuge for 12 minutes at 12000 rpm at 4C
- Remove supernatant and spin in vacuum centrifuge for 10 minutes
- Add 30 (20 for NTC) uL nfH2O to resuspend; pool samples A and B by reusing the water
Sample | ng/uL | ug in 30 uL |
Cells RCA Product (2AB,2BB) | 47.8 | 1.4 |
DEJ-CL-CellC01-20161116-Ind25