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==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 50ul 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)
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