Matt:LabNotes/2017-6-1

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Revision as of 20:30, 6 June 2017 by >Mzcai (→‎Conclusion)
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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

Prepare Beads

  1. 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
  2. Do 2 tubes, 1 for BarcodeV1 and 1 for BarcodeV3:
  3. Add 1ul Dynabeads MyOne Streptavidin C1 beads to 100ul buffer
  4. Vortex
  5. Pull down 1min on magnet
  6. Remove supernatant
  7. 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)
  8. Add to beads, pipette mix, and incubate 15min at room temp
  9. Pull down
  10. Wash twice with 100ul buffer (mix off magnet, and then pull down on magnet)
  11. 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. 
  1. Let gel slide dry in AirClean hood for 30min
  2. 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)
  3. Pipet 25ul onto center of gel
  4. Apply 18x30mm cover slip
  5. Apply an orange SecureSeal chamber
  6. Fill chamber with mineral oil and seal holes with stickies.
  7. Slide PCR on Biorad thermocycler
 94C 3min -> (94C 1min30sec -> 55C 1min -> 72C 2min) x 15 -> 72C 4min -> 4C hold
  1. 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...
  2. Put in 4C overnight

Strip Second Strand

  1. Remove SecureSeal and put slide in glass Coplin jar of Hexane for 5min
  2. Remove coverslip, wave slide to evaporate residual hexane, and scrape off residual adhesive
  3. Make Wash 1E: 10mM Tris + 50mM KCl + 2mM EDTA + 0.01% TX-100
  4. Shake slide in plastic box of Wash 1E twice for 4min
  5. Apply blue FrameSeal chamber
    • Hoping to use smaller volumes of Wash 1E
  6. Add ~800ul Wash 1E and then aspirate 2 times

Strip Second Strand

  1. Add 700ul 80% formamide in 2X SSC preheated to 80C and incubate 15min at room temp
  2. Wash twice with Wash 1E

Hybridize FISH probes

  1. Add 0.5uM dcProbe6-488 and 0.5uM dcProbe6-Cy5 in 30% formamide + 2X SSC preheated to 80C and incubate 10min at room temp
  2. Wash twice with 2X SSC
  3. Add 2X SSC and then seal with glass coverslip

Results

SYBR Green

File:20170604 w2 composite.jpg
File:20170605 w2 composite.jpg
File:20170606 w2 composite.jpg

FISH

  • Olympus Epifluorescence
    • Only saw signal 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:
    1. 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
    2. Image SYBR Green with and without beads
      • Image right after thermalcycler and then remove beads with magnet and image SYBR Green again
    3. Image after varying number of PCR cycles
      • 1 cycle, 15 cycles, 30 cycles
    4. To improve FISH, follow the Harvard Protocol more closely

Harvard Protocol

  1. Prepare 70% formamide 1X SSC in plastic coplin jar
    • 4mL 20X SSC
    • 56mL formamide
    • 20mL H2O
  2. Heat to 70C in microwave (~30sec in microwave) being careful not to let it boil over
  3. Put slides in and shake in 70C incubator 15min
  4. Transfer slides to separate coplin jar
  5. Wash in dH2O 3min on shaker
  6. Wash twice in Wash 1E 4min on shaker