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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 == {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> |- style="font-size:12pt" valign="bottom" | width="65" height="15" | 5uM |- style="font-size:12pt" valign="bottom" | height="15" | 100nM |- style="font-size:12pt" valign="bottom" | height="15" | 10nM |- style="font-size:12pt" valign="bottom" | height="15" | 1nM |- style="font-size:12pt" valign="bottom" | height="15" | 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 {| class="wikitable" <hiddentext>generated with [[:de:Wikipedia:Helferlein/VBA-Macro for EXCEL tableconversion]] V1.8<\hiddentext> |- style="font-size:12pt" align="center" | width="61" height="15" | Stock Tube | width="74" | Stock Volume | width="37" | 2XSSC | width="46" | Dilution | width="121" | Final concentration nM |- style="font-size:12pt" align="center" | align="center" height="15" | 100000 | align="center" | 7.5 | align="center" | 142.5 | align="center" | 0.05 | align="center" | 5000 |- style="font-size:12pt" align="center" | align="center" height="15" | 5000 | align="center" | 3 | align="center" | 147 | align="center" | 0.02 | align="center" | 100 |- style="font-size:12pt" align="center" | align="center" height="15" | 100 | align="center" | 15 | align="center" | 135 | align="center" | 0.1 | align="center" | 10 |- style="font-size:12pt" align="center" | align="center" height="15" | 10 | align="center" | 15 | align="center" | 135 | align="center" | 0.1 | align="center" | 1 |- style="font-size:12pt" align="center" | align="center" height="15" | 1 | align="center" | 15 | align="center" | 135 | align="center" | 0.1 | align="center" | 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 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) ==Protocol for stripping efficiency test==
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