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==Conclusion== *Initially thought the SYBR Green images looked good **The fluorescence signal was centered around beads and had diameter of ~8um after 15 cycles **Only a fraction of beads had fluorescence suggesting it was not autofluorescence and true polonies *Now after cleaning up fluorescent image and overlaying, it seems the fluorescence could be from the bead (maybe SYBR green binding to dsDNA on the bead) **Fluorescent polony size and BF bead size seem to match perfectly *No polony signal after FISH, could be due to bad FISH protocol or no polonies... *Areas to improve: *#Cleaner gel (there is too much crap on the gel) *#*Having a little debris is good for finding focus if there aren't any beads around *#Image SYBR Green with and without beads *#*Image right after thermalcycler and then remove beads with magnet and image SYBR Green again *#Image after varying number of PCR cycles *#*1 cycle, 15 cycles, 30 cycles *#To improve FISH, follow the Harvard Protocol more closely ===Harvard Protocol=== #Prepare 70% formamide 1X SSC in plastic coplin jar #*4mL 20X SSC #*56mL formamide #*20mL H2O #Heat to 70C in microwave (~30sec in microwave) being careful not to let it boil over #Put slides in and shake in 70C incubator 15min #Transfer slides to separate coplin jar #Wash in dH2O 3min on shaker #Wash twice in Wash 1E 4min on shaker
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