Matt:LabNotes/2017-6-8

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Image and Seq[edit]

Plan[edit]

  • PCR 3 gels and take them out at different cycles: 1, 15, and 30

Gel PCR[edit]

 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

Results[edit]

1 Cycle[edit]

  • 1um spots but not near beads
  • Beads can be seen drifting

File:20170608 1cycle.jpgFile:20170608 1cycle pos2.jpg

15 Cycles[edit]

  • Almost no signal except for on a couple beads

File:20170608 15cycle.jpgFile:20170608 15cycle pos2.jpg

30 Cycles[edit]

  • Large fraction (~50%) of beads have fluorescence

File:20170608 30cycle pos2.jpgFile:20170608 30cycle pos3.jpg

Conclusion[edit]

  • I don't know what the signal was in 1cycle samples
  • 15 and 30 cycles seems to have only amplified on beads
    • Hypothesis: the high number of primers/template strand on beads outcompetes the acrydite primers in gel
  • Dr. Zhang suggested adding 1/10th (50nM) of free floating Acrydite primer to help efficiency