Daniel:Notebook/ComboLock/2016-11-16: Difference between revisions
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>Djacobse |
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<li>qPCR</li> | <li>qPCR</li> | ||
</ol> | </ol> | ||
===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 2AC | |||
| 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 2BC | |||
| 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]])= | =Library Prep (Started [[Daniel:Notebook/ComboLock/2016-11-15|Yesterday]]; Exp. Start [[Daniel:Notebook/ComboLock/2016-11-7|11/7/16]])= |
Revision as of 20:48, 16 November 2016
BSA Beads Test (Started Yesterday)
Protocol
- 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
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 |
qPCR Sample Table
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 2AC | Dan | No BSA-biotin | D5 | 21 | 2 | 25 | 1 | 1 | 21 | 50 |
Sample 2BC | 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)
- 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 |