AlanFung:LabNotes/Rolony/2013-11-14: Difference between revisions
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100ul MALAT1 padlock probes in 2X ssc in various concentration | 100ul MALAT1 padlock probes in 2X ssc in various concentration | ||
*Original concentration 100uM, perform serial dilution | *Original concentration 100uM, perform serial dilution | ||
* | *{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> | ||
{| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> | |||
|- style="font-size:12pt" align="center" | |- style="font-size:12pt" align="center" | ||
| width="61" height="15" | Stock Tube | | width="61" height="15" | Stock Tube | ||
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MALAT1 padlock probes detection | |||
===Apparatus=== | ===Apparatus=== |
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
- {| 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
|}
MALAT1 padlock probes detection
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