AlanFung:LabNotes/Rolony/2013-11-14: Difference between revisions

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===Imaging===
===Imaging===
*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)

Revision as of 20:41, 19 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 (ppMALAT1)
  • Original concentration 100uM, perform serial dilution
Stock Tube Stock Volume 2XSSC 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 200ul to mattek dish
  • Incubate for 15 mins wash 3 times with 200ul pbs

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

  • Preheat 65C load

Imaging

  • 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)