AlanFung:LabNotes/Rolony/2013-11-14

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Revision as of 19:45, 19 November 2013 by >Alan6017518 (→‎Annealing)
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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

Image condition

  • 10-15um, 10 z stack image, lower limit begin go up till upper limit end
  • resolution (2kx2k)
  • 16bit,600Hz
  • when done save file (experiment_dish(mattekdate)_step#_dyeprobe_channel)

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 (ppMALAT1)
  • Original concentration 100uM, perform serial dilution
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
MALAT1 padlock probes detection
  • Dilute 110ul 10uM DC_Probe_2_Cy3 with 440ul 2X SSC
  • Shifted FISSEQ_2ndRCAprimerDye : 2nd Rolony from all padlock probes GGATATCGGGAAGCTGAA/3ATTO488N/

Apparatus

  • warm up incubator to 50C for incubation
  • warm up tube heater to 80C for pre-heating padlock probes

Stripping

  • Heat 80% formaide at 80C, add to mattek dish
  • Incubate for 10 mins wash with H2O twice

Annealing Padlock Probes

  • Pre-heat padlock probes at 80C for 5 mins
  • Add to cells at RT, and let it cool down over 5min

Annealing Universal Padlock Detection Probes

Imaging