AlanFung:LabNotes/Rolony/2013-11-14

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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

MALAT1 Padlock Probes Concentrations to test

5uM
100nM
10nM
1nM
0.1nM

Setup

  • In this experiment we will be using the 96well plate containing primary rolonies made on cy5 primary rolony detection probe

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

Protocol

Image and Count primary rolonies

  • Pick 6 positions and image the rolonies (Cy5)

Stripping

  • Heat 80% formaide at 80C, add ~50ul to 96 well plate
  • Incubate for 15 mins wash 3 times with 200ul pbs

Annealing Padlock Probes

  • Pre-heat padlock probes at 80C for 5 mins
  • aspirate
  • Add 100ul to cells at RT
  • transfer to 50C for 15 mins
  • aspirate and wash with pbs 3x

Annealing Universal Padlock Detection Probes

  • Preheat detection probes at 65C for 5 mins (2uM)
  • Add 100ul to cells at RT incubate for 15 mins
  • Wash with H2O twice and add 100ul 2x SSC

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)

Protocol for stripping efficiency test