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  
*
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MALAT1 padlock probes detection
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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

|- 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

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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

Imaging