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=Image and Seq= http://arep.med.harvard.edu/polony/polony_protocols/pcr.htm <br> http://arep.med.harvard.edu/polony/polony_protocols/sbe.htm *[[Matt:LabNotes/2017-6-14|Last time]] *Use new acrylamide primer (needs to be stored in -20C, aliquoted to avoid freeze/thaw) *Annealing temp of PCR changed from 55C -> 45C *One sample placed on magnet for 1min *One sample no magnet *K ==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 500nM (dsDNA PCR product with Uracil prepared by Dan 5.25.2017) #*BarcUv3 500nM (dsDNA PCR product with Uracil prepared by Dan 5.25.2017) #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 ==Cast 10% gels with primer== #Make Gel Mix #*12.5ul 199:1 40% A:B mix #*1ul 10% BSA #*33.5ul H2O #*1ul 50uM Acrydite primer #**Stored in -20C #*1ul 5% TEMED #*1ul 5% APS #Add 18ul to oval and cover with coverslip #Put slides in argon chamber and polymerize 30min #*Shake in H2O 30min ==Gel PCR== #Let gel slide dry in AirClean hood for 30min within 5 minutes of liquid film disappearance #Prepare 25ul PCR mix per gel #*2.5ul 5uM Primer2 #*12.5ul KAPA SYBR FAST MM #*5ul beads (1:1 mix of v1 and v3) #*1.25ul USER #*3.75ul H2O #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. #Place one slide on magnet for 1min #Slide PCR on Biorad thermocycler 37C 15min -> 94C 3min -> (94C 1min30sec -> 45C 1min -> 72C 2min) x 15 -> 72C 4min -> 4C hold #Place directly on Olympus with GFP/FITC filter ==Results== ===Control (no magnet)=== [[File:Composite_Control_Pos1.jpg|450px]][[File:Composite_Control_Pos2.jpg|450px]] ===Magnet=== [[File:Composite_Magnet_Pos1.jpg|450px]][[File:Composite_Magnet_Pos2.jpg|450px]] *Just GFP: [[File:Magnet_Pos2_GFP.jpg|450px]] ===Special Note=== *The z plane with fluorescence in focus is not the same as when beads are in focus *In these images, the in focus BF bead images was merged with in focus fluorescent images *What the BF channel looks like when the GFP channel is in focus: [[File:Composite_Magnet_Pos3.jpg|450px]] ==Conclusion== *Probably not rolonies, all fluorescent signal has similar characteristics that I have seen in previous tries: **Bright star shapes that look kind of like cell membrane stretched over cytoskeleton **Sometimes the star is surrounded by a fainter round signal *Do No Bead Control to see if crap is still here *Vary bead concentration *Dr. Zhang says 50-100um polonies is very normal size **Smaller template has larger polonies because it can diffuse easier *Do 50+ cycles overnight
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