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=Image and Seq= http://arep.med.harvard.edu/polony/polony_protocols/pcr.htm http://arep.med.harvard.edu/polony/polony_protocols/sbe.htm ==Cast 10% gels with primer== *Gels were cast [[Matt:LabNotes/2017-5-24]] and stored in water ==Prepare Beads== #Prepare 50ml Dynabeads buffer: 20ml 5M NaCl + 100ul 0.5M EDTA + 125ul 4M Tris-HCl + 29.775ml H2O #*2M NaCl, 1mM EDTA, 10mM Tris-HCl #Do 2 tubes, 1 for BarcodeV1 and 1 for BarcodeV3: #Add 1ul Dynabeads MyOne Streptavidin C1 beads to 100ul buffer #Vortex #Pull down 1min on magnet #Remove supernatant #Combined 15ul buffer with 15ul amplicon #*BarcUv1 240nM (dsDNA PCR product with Uracil prepared by Dan 5.5.2017) #*BarcUv3 220nM (dsDNA PCR product with Uracil prepared by Dan 5.5.2017) #Add to beads, pipette mix, and incubate 15min at room temp #Pull down #Wash twice with 100ul buffer (mix off magnet, and then pull down on magnet) #Bring volume to 40ul with H2O ==Gel PCR== Prepare diffuse-in mix while allowing slides to dry. You don't want the slides to over-dry, though. You will observe a thin, shrinking film of liquid on the surface of each gel. Generally, we try to add the diffuse-in mix within 5 minutes or so of this shrinking film's disappearance. This generally means about 30 minutes of drying. #Let gel slide dry in AirClean hood for 30min #Prepare 25ul PCR mix per gel #*2.5ul 5uM Primer2 #*12.5ul KAPA SYBR FAST MM #*10ul beads (1:1 mix of v1 and v3) #Pipet 25ul onto center of gel #Apply 18x30mm cover slip #Apply an orange SecureSeal chamber #Fill chamber with mineral oil and seal holes with stickies. #Slide PCR on Biorad thermocycler 94C 3min -> (94C 1min30sec -> 55C 1min -> 72C 2min) x 15 -> 72C 4min -> 4C hold #Place directly on Olympus with GFP/FITC filter #*Saw polonies! #**Approx 8um in diameter and centered around the beads #**Confident not autofluorescence from the bead because only ~1/4 beads had a polony #**The 1/4 polony success rate might need to be improved but right now it may just be due to beads with no DNA #**Edit: might not be polony if SYBR Green stained dsDNA on the bead... #Put in 4C overnight ==Strip Second Strand== #Remove SecureSeal and put slide in glass Coplin jar of Hexane for 5min #Remove coverslip, wave slide to evaporate residual hexane, and scrape off residual adhesive #Make Wash 1E: 10mM Tris + 50mM KCl + 2mM EDTA + 0.01% TX-100 #Shake slide in plastic box of Wash 1E twice for 4min #Apply blue FrameSeal chamber #*Hoping to use smaller volumes of Wash 1E #Add ~800ul Wash 1E and then aspirate 2 times ==Strip Second Strand== #Add 700ul 80% formamide in 2X SSC preheated to 80C and incubate 15min at room temp #Wash twice with Wash 1E ==Hybridize FISH probes== #Add 0.5uM dcProbe6-488 and 0.5uM dcProbe6-Cy5 in 30% formamide + 2X SSC preheated to 80C and incubate 10min at room temp #Wash twice with 2X SSC #Add 2X SSC and then seal with glass coverslip ==Results== ===SYBR Green=== [[File:20170604_w2_composite.jpg|650px]]<br> [[File:20170605_w2_composite.jpg|650px]]<br> [[File:20170606_w2_composite.jpg|650px]]<br> ===FISH=== [[File:20170607_w1_composite.jpg|650px]] *Olympus Epifluorescence (GFP in green, Cy5 in red) **Only saw round signal that could be polony in GFP channel but looked like beads autofluorescence (could also be FISH probes on beads) **No signal in Cy5 channel (tried exposure time from 100ms to 1s) *Magnetic beads were still on the gel, often much more clumped together *No signal under confocal ==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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