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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/2017-6-27|Last time]] *This time 3 samples: **No beads **1ul beads **5ul beads ==Prepare Beads== [[Matt:LabNotes/2017-6-27#Prepare_Beads|Beads prepared 2 weeks ago]] ==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 #Slide1 #*2.5ul 5uM Primer2 #*12.5ul KAPA SYBR FAST MM #*1.25ul USER #*8.75ul H2O #Slide2 #*2.5ul 5uM Primer2 #*12.5ul KAPA SYBR FAST MM #*1ul beads (1:1 mix of v1 and v3) #*1.25ul USER #*7.75ul H2O #Slide3 #*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 30min -> 94C 3min -> (94C 45sec -> 45C 1min -> 72C 2min) x 50 -> 72C 4min -> 4C hold #Place directly on Olympus with GFP/FITC filter ==Results== ===Slide1 (no beads)=== [[File:20170713_NoBeads_Slide1_Pos1_w2GFP.jpg|350px]][[File:20170713_NoBeads_Slide1_Pos2_w2GFP.jpg|350px]]<br> [[File:20170713_NoBeads_Slide1_Pos3_w2GFP.jpg|350px]][[File:20170713_NoBeads_Slide1_Pos4_w2GFP.jpg|350px]] ===Slide2 (few beads)=== [[File:20170713_1ulBeads_Slide2_Pos1_w2GFP.jpg|350px]][[File:20170713_1ulBeads_Slide2_Pos2_w2GFP.jpg|350px]] ===Slide3 (many beads)=== [[File:20170713_5ulBeads_Slide3_Pos1_w2GFP.jpg|350px]][[File:20170713_5ulBeads_Slide3_Pos2_w2GFP.jpg|350px]] ==Conclusion== *Seems like all signal that could be interpreted as polonies is also present in Slide1 (no beads negative control). *Slide2 first image has 2 20um diameter bright round spots that could be polonies and didn't find any similar spot in negative control slide **Need to do FISH to confirm ***FISH determined there are no polonies *NEXT STEP: **Try with reverse complement primers: AcryditePrimer2 and Primer1 **Try with Jumpstart Taq polymerase and no SYBR green in pcr mix ***Follow Harvard protocol exactly: BSA and Tween-20 ***Stain with SYBR gold or directly do FISH =Hybridization to Confirm= *On 7-15-2017 do "Slide clean up" from [http://arep.med.harvard.edu/polony/polony_protocols/pcr.htm] *Store in Wash 1E Buffer at 4C *Apply blue FrameSeal chamber *Strip with 80% formamide in 2X SSC twice **Preheat to 75C and then add 500ul to slide and incubate at 55C for 15min **Wash with Wash 1E twice *Image to see if crap is washed away **Nope looked exactly the same under GFP filter **No beads in BF image *Hybridize dcProbe6-488 and dcProbe6-Cy5 **Preheat 0.5uM dcProbe6-488 and 0.5uM dcProbe6-Cy5 in 30% formamide + 2X SSC to 75C **Add 100ul to each slide and incubate 10min at RT **Wash with 2X SSC twice *Image **GFP channel looked exactly the same as previously **Nothing seen in Cy5 channel
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