Matt:LabNotes/2017-6-8

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Revision as of 01:53, 9 June 2017 by >Mzcai (Created page with "=Image and Seq= *Last time ==Plan== *PCR 3 gels and take them out at different cycles: 1, 15, and 30 ==Gel PCR== Prepare diffuse-in m...")
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Image and Seq

Plan

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

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