AlanFung:LabNotes/Rolony/2013-11-14

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Optimum Probe# Test[edit]

  • 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[edit]

5uM
100nM
10nM
1nM
0.1nM

Setup[edit]

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

Reagents[edit]

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[edit]

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

Protocol[edit]

Image and Count primary rolonies[edit]

  • Pick 6 positions and image the rolonies (Cy5)

Stripping[edit]

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

Annealing Padlock Probes[edit]

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

Annealing Universal Padlock Detection Probes[edit]

  • 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[edit]

  • 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[edit]