Daniel:Notebook/ComboLock/2016-8-26: Difference between revisions
>Djacobse (Created page with "=Positive Control Amplicon Test 2 (Started Monday)= Back to Calendar ==Protocol-Library Prep== <ol sta...") |
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<li>Ethanol Precipitation</li> | <li>Ethanol Precipitation (from [[Daniel:Notebook/ComboLock/2016-8-25|yesterday]])</li> | ||
<ol type="A" start="3"> | <ol type="A" start="3"> | ||
<li>Centrifuge at 12000rpm at 4C for 25 minutes</li> | <li>Centrifuge at 12000rpm at 4C for 25 minutes</li> | ||
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|- style="font-size:12pt" | |- style="font-size:12pt" | ||
| height="15" valign="bottom" | 20160822 Sample A | | height="15" valign="bottom" | 20160822 Sample A | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 38.4 | ||
|- style="background-color:#D9D9D9;font-size:12pt" | |- style="background-color:#D9D9D9;font-size:12pt" | ||
| height="15" valign="bottom" | 20160822 Sample B | | height="15" valign="bottom" | 20160822 Sample B | ||
| align="center" align="center" valign="bottom" | | | align="center" align="center" valign="bottom" | 30.1 | ||
|} | |} | ||
=Positive Control Dilution Test= | |||
The idea behind this is that the positive controls I have been using have been at levels of about 1 pmol, which would be approximately 10^11 molecules available for latch/padlock binding. This might be an issue, since the experiments I've been running have fewer available molecules. For example, 10,000 cells at 10,000 transcripts per cell (about as much as would ever exist) is still only 10^8 copies for detection, and that is assuming ALL detection probes (C probes or antibodies) have bound in pairs, which is unlikely. More reasonable copy numbers would be 10^7 (~thousand/cell), 10^6 (~hundred/cell) or even 10^5 (~ten/cell) for some. I will try to go as low as possible and see where the detection ends. | |||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8</hiddentext> | |||
|- style="background-color:#B7DEE8;font-size:12pt;font-weight:bold" align="center" | |||
| width="65" height="27" | Tube No | |||
| width="83" | Dilution | |||
| width="68" | Molecules Available | |||
|- style="font-size:12pt" | |||
| align="center" height="15" valign="bottom" | 0 | |||
| align="center" align="center" valign="bottom" | 1 | |||
| align="center" align="center" valign="bottom" | 100000000000 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| align="center" height="15" valign="bottom" | 1 | |||
| align="center" align="center" valign="bottom" | 10 | |||
| align="center" align="center" valign="bottom" | 10000000000 | |||
|- style="font-size:12pt" | |||
| align="center" height="15" valign="bottom" | 2 | |||
| align="center" align="center" valign="bottom" | 100 | |||
| align="center" align="center" valign="bottom" | 1.E+09 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| align="center" height="15" valign="bottom" | 3 | |||
| align="center" align="center" valign="bottom" | 1000 | |||
| align="center" align="center" valign="bottom" | 1.E+08 | |||
|- style="font-size:12pt" | |||
| align="center" height="15" valign="bottom" | 4 | |||
| align="center" align="center" valign="bottom" | 10000 | |||
| align="center" align="center" valign="bottom" | 1.E+07 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| align="center" height="15" valign="bottom" | 5 | |||
| align="center" align="center" valign="bottom" | 100000 | |||
| align="center" align="center" valign="bottom" | 1.E+06 | |||
|- style="font-size:12pt" | |||
| align="center" height="15" valign="bottom" | 6 | |||
| align="center" align="center" valign="bottom" | 1.E+06 | |||
| align="center" align="center" valign="bottom" | 1.E+05 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| align="center" height="15" valign="bottom" | 7 | |||
| align="center" align="center" valign="bottom" | 1.E+07 | |||
| align="center" align="center" valign="bottom" | 10000 | |||
|- style="font-size:12pt" | |||
| align="center" height="15" valign="bottom" | 8 | |||
| align="center" align="center" valign="bottom" | 1.E+08 | |||
| align="center" align="center" valign="bottom" | 1000 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| align="center" height="15" valign="bottom" | 9 | |||
| align="center" align="center" valign="bottom" | 1.E+09 | |||
| align="center" align="center" valign="bottom" | 100 | |||
|- style="font-size:12pt" | |||
| align="center" height="15" valign="bottom" | 10 | |||
| align="center" align="center" valign="bottom" | 1.E+10 | |||
| align="center" align="center" valign="bottom" | 10 | |||
|- style="background-color:#D9D9D9;font-size:12pt" | |||
| align="center" height="15" valign="bottom" | 11 | |||
| align="center" align="center" valign="bottom" | 1.E+11 | |||
| align="center" align="center" valign="bottom" | 1 | |||
|} | |||
I'll make the dilutions by diluting 5 uL of the previous dilution into 45 uL nfH2O. Hopefully this will be enough to prevent dilution artifacts until we get down near the lower limits. | |||
Since it is late in the afternoon already, I'll make the dilutions today and begin the experiment [[Daniel:Notebook/ComboLock/2016-8-27|tomorrow]]. | |||
[[Category:ComboLock]] [[Category:20160826]] |
Latest revision as of 23:21, 26 August 2016
Positive Control Amplicon Test 2 (Started Monday)[edit]
Protocol-Library Prep[edit]
- Ethanol Precipitation (from yesterday)
- Centrifuge at 12000rpm at 4C for 25 minutes
- Remove supernatant and add 750 uL 70% EtOH
- Centrifuge at 12000rpm at 4C for 15 minutes
- Remove supernatant and let dry in PCR hood for 15 min
- Resuspend in 30 uL nfH2O
Nanodrop Results[edit]
Sample | ng/uL ssDNA |
20160822 Sample A | 38.4 |
20160822 Sample B | 30.1 |
Positive Control Dilution Test[edit]
The idea behind this is that the positive controls I have been using have been at levels of about 1 pmol, which would be approximately 10^11 molecules available for latch/padlock binding. This might be an issue, since the experiments I've been running have fewer available molecules. For example, 10,000 cells at 10,000 transcripts per cell (about as much as would ever exist) is still only 10^8 copies for detection, and that is assuming ALL detection probes (C probes or antibodies) have bound in pairs, which is unlikely. More reasonable copy numbers would be 10^7 (~thousand/cell), 10^6 (~hundred/cell) or even 10^5 (~ten/cell) for some. I will try to go as low as possible and see where the detection ends.
Tube No | Dilution | Molecules Available |
0 | 1 | 100000000000 |
1 | 10 | 10000000000 |
2 | 100 | 1.E+09 |
3 | 1000 | 1.E+08 |
4 | 10000 | 1.E+07 |
5 | 100000 | 1.E+06 |
6 | 1.E+06 | 1.E+05 |
7 | 1.E+07 | 10000 |
8 | 1.E+08 | 1000 |
9 | 1.E+09 | 100 |
10 | 1.E+10 | 10 |
11 | 1.E+11 | 1 |
I'll make the dilutions by diluting 5 uL of the previous dilution into 45 uL nfH2O. Hopefully this will be enough to prevent dilution artifacts until we get down near the lower limits.
Since it is late in the afternoon already, I'll make the dilutions today and begin the experiment tomorrow.