Matt:LabNotes/2017-6-13

From ZhangLabWiki
Revision as of 19:30, 13 June 2017 by >Mzcai (Created page with "=Image and Seq= *Matt:LabNotes/2017-6-8 confirmed no polonies **Ideas for why: ***Gels were cast and stored poorly in water (UV slides beforehand, use immediate...")
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)
Jump to navigation Jump to search

Image and Seq

  • Matt:LabNotes/2017-6-8 confirmed no polonies
    • Ideas for why:
      • Gels were cast and stored poorly in water (UV slides beforehand, use immediately or store in buffer with EDTA)
      • Primers on beads outcompeted primers in gel (USER digest amplicons on beads to shorten primer on beads)

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

Sample Conditions

  • 3 samples to test how USER can help create polonies
    • USER will nick the biotinylated strand leaving only: CAGTGT

Samples:

  1. Basic repeat of last time with fresh gels
  2. USER digest beads before adding to gel
  3. Include USER enzyme in the PCR mix

Cast 10% gels with primer

  1. Make Gel Mix
    • 25ul 199:1 40% A:B mix
    • 2ul 10% BSA
    • 67ul H2O
    • 2ul 50uM Acrydite primer
    • 2ul 5% TEMED
    • 2ul 5% APS
  2. Add 18ul to oval and cover with coverslip
  3. Put slides in argon chamber and polymerize 30min
  4. Wash in H2O 30min shaker

Prepare Beads

  1. Beads prepared on Matt:LabNotes/2017-5-24

=For USER prenicked Beads (Sample 2)

  • Take 10ul beads (1:1 mix of v1 and v3)
  • Add 5ul 10X Cutsmart Buffer
  • Add 5ul USER
  • Add 30ul H2O
  • Incubate 15min @ 37C
  • Pull down beads, remove supernatant, and resuspend in 10ul Dynabeads buffer

Gel PCR

  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
    • 10ul beads (1:1 mix of v1 and v3)
    • For USER sample 3 include 1.25ul USER and use 1.25ul 10uM Primer2 instead
  3. Pipet 25ul onto center of gel
  1. Apply 18x30mm cover slip
  2. Apply an orange SecureSeal chamber
  3. Fill chamber with mineral oil and seal holes with stickies.
  4. Slide PCR on Biorad thermocycler
    • Put in sample 3 first, put in sample 1 & 2 after 37C 30min incubation
 37C 30min -> 94C 3min -> (94C 1min30sec -> 55C 1min -> 72C 2min) x 15 -> 72C 4min -> 4C hold
  1. Place directly on Olympus with GFP/FITC filter