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==Motor Neurons RNA FISH Dye Coupling== *[[Matt:LabNotes/2015-6-8#RNA_FISH | Previous RNA FISH attempt detected no signal]] **Try again with higher expressed genes (CUX2 and KIT) *[[Matt:LabNotes/2015-6-18 | RNA FISH continued here]] ===Probe Resuspension=== **[[Media:GAO_022315.xlsx | Table of ordered plates]] ***KIT: Plate=KIT-SNAP25-20150128 Wells=A1-D12 ***SNAP25: Plate=KIT-SNAP25-20150128 Wells=E1-H12 #Resuspend each oligo in 15 uL nuclase-free H2O (1 mM) #Take 2 uL from each oligo well and combine them in a single 1.5 mL tube (total concentration 1 mM) ##Store the remaining oligos at -20C #Add 450 uL nuclase-free, diluting 10:1, for final concentration of 100 uM #*Technically closer to 5:1 dilution but final result is closer to 10:1 because starting concentration is actually lower... #*Goal is to get 1ug/ul DNA concentration #Check concentration in nanodrop at 1:1, 5:1, and 10:1 dilutions for accuracy {| {{table}} | align="center" style="background:#f0f0f0;"|'''''' | align="center" style="background:#f0f0f0;"|'''measured (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''normalized (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''Average''' | align="center" style="background:#f0f0f0;"|'''Stdev''' |- | KIT 1:1||1053.5||1053.5|||| |- | KIT 1:5||212.5||1062.5|||| |- | KIT 1:10||104.9||1049||1055||6.87 |- | SNAP25 1:1||1039.5||1039.5|||| |- | SNAP25 1:5||207.7||1038.5|||| |- | SNAP25 1:10||107.4||1074||1050.67||20.21 |- |} ===Dye Coupling=== [[Daniel:Notebook/RNAFACS/2015-4-10#Dye_Labeling | Dan's best practice dye coupling protocol]]<br> [[Daniel:Protocols/AlexaDyeCouple#ARES | Generic dye coupling protocol]] *Sebastian said in email the two highest expressed genes from our 48 probe set of genes are CUX2 and KIT *I am going to try both 488/594 and 546/647 pairs of fluorophores in case one set is not compatible with our Olympus filter cubes CUX2-488 KIT-594 CUX2-546 KIT-647 #Warm DMSO and bicarbonate labeling buffer and nuclease free H2O to room temperature #Add 5 uL DNA (~1ug/ul) and 3 uL of sodium bicarbonate buffer #Denature samples for 5 minutes at 95C, then snap cool using ice box #Dissolve 1 vial of reactive dye in 2 uL DMSO; do multiple cycles of vortex and spin down during 5 minutes of denaturing #*Dye cannot be saved for later use. Use immediately! #Add 8 uL sample to dye tube #Incubate in the dark for 1 hour #Add 10 uL 3M NaOAc and 80 uL 1xTE to sample #[[Daniel:Protocols/CentriSep|Centri-Sep]] column purification after incubation #*Use 1X TE Buffer #*No vacufuge #Ethanol Precipitation; add 250 uL 100% EtOH, 10 uL NaOAc and 0.5 uL glycoblue; incubate overnight #Centrifuge at 4 C for 30 minutes (14,000 rpm) #Remove supernatant and add 750 uL chilled 75% EtOH #Centrifuge 5 minutes at 4 C #Dry the pellet in the hood #Resuspend pellet in 12 uL TE #Check dye:probe ratio with Nanodrop #*Use 324.5 g/mol/bp as molar mass of ssDNA (6490 g/mol of 20-mer) #*Starting DNA mass = 5ul * 1ug/ul {| {{table}} | align="center" style="background:#f0f0f0;"|'''Probe''' | align="center" style="background:#f0f0f0;"|'''ssDNA (ng/ul)''' | align="center" style="background:#f0f0f0;"|'''488 dye (pmol/ul)''' | align="center" style="background:#f0f0f0;"|'''594 dye (pmol/ul)''' | align="center" style="background:#f0f0f0;"|'''546 dye (pmol/ul)''' | align="center" style="background:#f0f0f0;"|'''647 dye (pmol/ul)''' | align="center" style="background:#f0f0f0;"|'''dye:probe ratio''' | align="center" style="background:#f0f0f0;"|'''ssDNA mass (ug)''' | align="center" style="background:#f0f0f0;"|'''ssDNA start mass (ug)''' | align="center" style="background:#f0f0f0;"|'''ssDNA Yield (%)''' |- | CUX2-488||227.7||39.1||0||N/A||N/A||1.11||2.7324||5||54.648 |- | KIT-594||251.2||-0.6||51.6||N/A||N/A||1.33||3.0144||5||60.288 |- | CUX2-546||246.9||N/A||N/A||43.7||0.2||1.15||2.9628||5||59.256 |- | KIT-647||268.6||N/A||N/A||6.8||45.6||1.10||3.2232||5||64.464 |} *[[Matt:LabNotes/2015-6-18 | RNA FISH continued here]]
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