Matt:LabNotes/2017-6-27

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Image and Seq[edit]

http://arep.med.harvard.edu/polony/polony_protocols/pcr.htm
http://arep.med.harvard.edu/polony/polony_protocols/sbe.htm

  • 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[edit]

  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 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)
  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

Cast 10% gels with primer[edit]

  1. 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
  2. Add 18ul to oval and cover with coverslip
  3. Put slides in argon chamber and polymerize 30min
    • Shake in H2O 30min

Gel PCR[edit]

  1. Let gel slide dry in AirClean hood for 30min within 5 minutes of liquid film disappearance
  2. 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
  1. Pipet 25ul onto center of gel
  2. Apply 18x30mm cover slip
  3. Apply an orange SecureSeal chamber
  4. Fill chamber with mineral oil and seal holes with stickies.
  5. Place one slide on magnet for 1min
  6. Slide PCR on Biorad thermocycler
 37C 15min -> 94C 3min -> (94C 1min30sec -> 45C 1min -> 72C 2min) x 15 -> 72C 4min -> 4C hold
  1. Place directly on Olympus with GFP/FITC filter

Results[edit]

Control (no magnet)[edit]

File:Composite Control Pos1.jpgFile:Composite Control Pos2.jpg

Magnet[edit]

File:Composite Magnet Pos1.jpgFile:Composite Magnet Pos2.jpg

Special Note[edit]

  • 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

Conclusion[edit]

  • 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