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===Exo I/III in situ to check 2ndary Rolony Ampligase reaction=== *Currently 2nd Rolony fabrication is inefficient or unsuccessful **Need a positive and negative control (currently working on making positive control by pre-circularizing ppMALAT1) **Trying to determine whether Ampligase reaction or 2nd RCA is the limiting step *How to separate Ampligase step from 2nd RCA step? **Add Exo I/III to cells after Ampligase step to digest non-circular DNA **Then can use Shifted FISSEQ_2ndRCAprimerDye to detect the padlock probes that are left (and therefore circularized) **By using positive and negative control, can determine yield of Ampligase step ====Procedure==== *Use 6 wells with 1st Rolonies from 96-well plate: *2 Positive control (C7 & D7): +Ampligase, -Exo (expect to see lots of signal like previous experiments) *2 Negative control (E7 & F7): -Ampligase, +Exo (expect to see no signal) *2 Experimental (D9 & E9): +Ampligase, +Exo (expect to see a % of a positive control, hopefully close to 100%) '''Determine Number of ppMALAT1 targets''' #Hybridize 2nM ppMALAT1 (in 2X SSC) ##Preheat to 70C for 5min and then add 50ul to each well ##Incubate 50C for 10min ##Wash once with 2X SSC #Add 2uM Shifted FISSEQ_2ndRCAprimerDye in 30% formamide and 2X SSC (206.5ul H2O + 105ul 100% formamide + 3.5ul 200uM Dye + 35ul 20X SSC = 350ul) ##Preheat to 60C for 5min and then add 50ul to each well ##Incubate RT for 15min ##Wash 2 times with 2X SSC #Image (ATTO488) #Strip with 80% formamide for 10min @50C '''Ampligase Experiment''' #Hybridize 2nM ppMALAT1 (in 2X SSC) ##Preheat to 70C for 5min and then add 50ul to each well ##Incubate 50C for 10min ##Wash once with 2X SSC #Add 0.05U/ul Ampligase to Positive and Exprimental (2ul Ampligase + 20ul 10X Buffer + 178ul H2O) ##Negative control: (90ul H2O + 10ul 10X buffer) ##Incubate 50C for 4hrs #Wash twice with 1X PBS #Add 50ul Exo I/III to each well (2.5ul 20U/ul Exo I and 2.5ul 200U/ul Exo III) ##Exo I/III mix: 20ul 10X Exo III Buffer + 10ul Exo I + 10ul Exo III + 160ul H2O ##Positive control: 50ul of (10ul 10X Exo III Buffer + 90ul H2O) ##Incubate 37C for 2hrs #Wash twice with 1X PBS preheated to 94C ##Let sit for 1min before aspirating #Add 2uM Shifted FISSEQ_2ndRCAprimerDye in 30% formamide and 2X SSC (206.5ul H2O + 105ul 100% formamide + 3.5ul 200uM Dye + 35ul 20X SSC = 350ul) ##Preheat to 60C for 5min and then add 50ul to each well ##Incubate RT for 15min ##Wash 2 times with 2X SSC #Image (ATTO488)
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