AlanFung:LabNotes/Rolony/2013-11-14: Difference between revisions
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80% Formamide in 2X SSC | 80% Formamide in 2X SSC | ||
* | * | ||
MALAT1 padlock probes | 100ul MALAT1 padlock probes in 2X ssc in various concentration | ||
*Original concentration 100uM, perform serial dilution | |||
* | |||
MALAT1 padlock probes detection | MALAT1 padlock probes detection | ||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> | |||
|- style="font-size:12pt" align="center" | |||
| width="61" height="15" | Stock Tube | |||
| width="74" | Stock Volume | |||
| width="37" | SSC | |||
| width="46" | Dilution | |||
| width="121" | Final concentration nM | |||
|- style="font-size:12pt" align="center" | |||
| align="center" height="15" | 100000 | |||
| align="center" | 7.5 | |||
| align="center" | 142.5 | |||
| align="center" | 0.05 | |||
| align="center" | 5000 | |||
|- style="font-size:12pt" align="center" | |||
| align="center" height="15" | 5000 | |||
| align="center" | 3 | |||
| align="center" | 147 | |||
| align="center" | 0.02 | |||
| align="center" | 100 | |||
|- style="font-size:12pt" align="center" | |||
| align="center" height="15" | 100 | |||
| align="center" | 15 | |||
| align="center" | 135 | |||
| align="center" | 0.1 | |||
| align="center" | 10 | |||
|- style="font-size:12pt" align="center" | |||
| align="center" height="15" | 10 | |||
| align="center" | 15 | |||
| align="center" | 135 | |||
| align="center" | 0.1 | |||
| align="center" | 1 | |||
|- style="font-size:12pt" align="center" | |||
| align="center" height="15" | 1 | |||
| align="center" | 15 | |||
| align="center" | 135 | |||
| align="center" | 0.1 | |||
| align="center" | 0.1 | |||
|} | |||
===Apparatus=== | ===Apparatus=== | ||
*warm up incubator to 50C for incubation | *warm up incubator to 50C for incubation |
Revision as of 21:54, 14 November 2013
Optimum Probe# Test
- In this experiment we are trying to find the concentration of probes that works best for capturing rolony targets. From previous experiments we know that a concentration of 0.6uM with a volume of 100ul works.
- A test of 5 different concentrations will be perform and image analysis using PISA will be performed to count the number of target being captured
MALAT1 Padlock Probes Concentrations to test
5uM |
100nM |
10nM |
1nM |
0.1nM |
Protocol
- In this experiment we will be using the Mattek dish containing primary rolonies made on Oct 23rd dish #2
Reagents
80% Formamide in 2X SSC
100ul MALAT1 padlock probes in 2X ssc in various concentration
- Original concentration 100uM, perform serial dilution
MALAT1 padlock probes detection
Stock Tube | Stock Volume | SSC | Dilution | Final concentration nM |
100000 | 7.5 | 142.5 | 0.05 | 5000 |
5000 | 3 | 147 | 0.02 | 100 |
100 | 15 | 135 | 0.1 | 10 |
10 | 15 | 135 | 0.1 | 1 |
1 | 15 | 135 | 0.1 | 0.1 |
Apparatus
- warm up incubator to 50C for incubation
- warm up tube heater to 80C for pre-heating padlock probes
Stripping
Annealing
- Pre-heat padlock probes at 80C for 5 mins