Matt:LabNotes/2017-6-13

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Image and Seq[edit]

  • Last time 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[edit]

  • 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[edit]

  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[edit]

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

For USER prenicked Beads (Sample 2)[edit]

  • Take 5ul beads (1:1 mix of v1 and v3)
  • Add 2.5ul 10X Cutsmart Buffer
  • Add 2.5ul USER
  • Add 15ul H2O
  • Incubate 15min @ 37C
  • Put on cold block 5min
  • Pull down beads, remove supernatant, and resuspend in 5ul Dynabeads buffer
    • Was not able to pull down beads so just pipetted the reaction mix (1/5 diluted beads)

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)
    • 5ul H2O
    • For USER sample 3 include 1.25ul USER and use 3.75ul H2O 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 15min -> 94C 3min -> (94C 1min30sec -> 55C 1min -> 72C 2min) x 20 -> 72C 4min -> 4C hold
  • Made a mistake and did 15sec 37C at first, didn't catch the mistake until after 94C 3min incubation probably killing the USER enzyme
    • Restarted the thermalcycle program anyway just in case but probably won't work
  1. Place directly on Olympus with GFP/FITC filter

Results[edit]

Sample 1: Standard[edit]

  • Like seen before, amplification on beads only

File:20160613 Sample1 Standard composite.jpg

Samples 2: Prenick with USER[edit]

  • Signal only in beads

File:20160613 Sample2 PreUSER2 composite.jpgFile:20160613 Sample2 PreUSER5 composite.jpg

Sample 3: USER in PCR[edit]

  • Again signal only in beads
  • Expected because not enough time for USER digest

File:20160613 Sample3 USER composite.jpgFile:20160613 Sample3 USER1 composite.jpg450px

Conclusion[edit]

  • Need to try USER in PCR again
  • Try adding 1/10th free floating acrydite primer
  • Try following Harvard protocol more closely
  • Fresh gels made no difference