Matt:LabNotes/2017-6-14

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Image and Seq[edit]

  • Last time
    • messed up the sample with USER in PCR mix
    • pre-nicking with USER did not work

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:

  1. Include 50nM Acrydite primer
  2. Include USER enzyme in the PCR mix
    • USER will nick the biotinylated strand leaving only: CAGTGT
  3. Include 0.1% Tween-20 and 0.2% BSA
    • These are included in the Harvard protocol

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

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 Sample1
    • 2.5ul 5uM Primer2
    • 1.25ul 1uM Acrydite Primer (50nM final)
    • 12.5ul KAPA SYBR FAST MM
    • 5ul beads (1:1 mix of v1 and v3)
    • 3.75ul H2O
  3. Prepare 25ul PCR mix Sample2
    • 2.5ul 5uM Primer2
    • 12.5ul KAPA SYBR FAST MM
    • 1.25ul USER
    • 5ul beads (1:1 mix of v1 and v3)
    • 3.75ul H2O
  4. Prepare 25ul PCR mix Sample3
    • 2.5ul 5uM Primer2
    • 12.5ul KAPA SYBR FAST MM
    • 5ul beads (1:1 mix of v1 and v3)
    • 2.5ul 1% Tween-20
    • 2.5ul 2% BSA
  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. Slide PCR on Biorad thermocycler
    • Put in sample 2 first, put in sample 1 & 3 after 37C 30min incubation
 37C 30min -> 94C 3min -> (94C 1min30sec -> 55C 1min -> 72C 2min) x 20 -> 72C 4min -> 4C hold
  1. Place directly on Olympus with GFP/FITC filter

Results[edit]

  • All 3 had deformed SecureSeals and were leaking oil
  • Used 20X objective to image

Sample 1[edit]

File:20170614 Sample1 2ndprimer composite.jpg

Sample 2[edit]

  • Spots about 4um in diameter

File:20170614 Sample2 USER composite.jpgFile:20170614 Sample2 USER1 composite.jpg

Sample 3[edit]

File:20170614 Sample3 Tween20 composite.jpg

Conclusion[edit]

  • Sample 2 with USER looks promising but could also just be signal similar to 1 cycle
  • Sample 1 and 3 are not polonies
  • Did some research into acryloyl groups and acrydite-modified primers. Very unstable and should not be stored at 4C like I have been. Order new acrydite-primers and aliquot, store at -20C
  • Tm of Primer2 is 53C... so try decreasing annealing temp of PCR to 45C