Team:Illinois/Antibody GPCR Fusion Notebook

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Contents

Recipes

  • Tris-Cl, 1M
    • Dissolve 121g Tris base in 800ml H2O
    • Adjust to desired pH with concentrated HCl
    • Mix and add H2O to 1 liter
    • (Approximately, 20ml HCl for pH 7.4 and 42ml for pH 8.0)


  • EDTA, 0.5M (pH 8.0)
    • Dissolve 186.1g Na2 EDTA-2H2O in 700ml H20
    • Adjust pH to 8.0 with 10M NaOH(~50ml)
    • Add H2O to 1 liter


  • Breaking buffer - 100ml
    • 2ml Triton X-100
    • 1ml Sodium dodecyl sulfate (SDS)
    • 0.5844g NaCl (100mM)
    • 1ml 1M Tris-Cl pH 8.0 (10mM)
    • 200uL 0.5M EDTA (1mM)


22nd July

  • Yeast obtained from Dr. Zhao


24th July

  • Prepared liquid culture for DNA extraction
  • Made 1M Tris. Cl pH 8.0
  • Made 4M ammonium acetate


22nd August

  • Attempted DNA extraction
    • Result: Failed
  • Obtained more yeast from Dr. Zhao


25th August

  • Prepared overnight culture for DNA extraction (3:27pm)


26th August

  • Attempted DNA extraction
  • Prepped overnight culture


27th August

  • Performed PCR: PGK Terminator
Buffer G 12.5uL x4 50uL
Forward Primer 0.5uL x4 2uL
Reverse Primer 0.5uL x4 2uL
H2O 10.8uL x4 43.2uL
Taq 0.2uL x4 0.8uL
template 0.5ul
Negative control 3 H2O


  • PCR program:
    • 4 min 94 degrees
    • 25-30x 30s 94 degrees
    • 30s Tm primers
    • 1 min/KB 72 degrees
    • 7 min 72 degrees
  • For the gel: 5uL loading dye gel is in cold room
  • Prepped 3 overnight cultures

28th August

  • Extracted DNA from 4 cultures
  • Ran gel of PCR products (1.5%, 200V)
    • Result: No bands present


2nd September

  • PCR: PGK Terminator
Mastermix 10uL x3 30uL
Forward Primer 0.5uL x3 1.5uL
Reverse Primer 0.5uL x3 1.5uL
Template 10uL x3 30uL
H2O 10.8uL x4 43.2uL
Negative control 25uL H2O

3rd September

  • Ran gel
    • Ladder lane 7
    • Sample 7 spilled
    • 1% agarose
      • Too high
    • 120V
      • Too low
    • 50 minutes


8th September

  • PCR: PGK Terminator
Mastermix 10uL x3 30uL
Forward Primer 0.5uL x3 1.5uL
Reverse Primer 0.5uL x3 1.5uL
Template 14uL x3 42uL
  • Prepped 4 overnight cultures
    • Yeast dried out again

9th September

  • Signs of life in 3 of the cultures
    • Wait until tomorrow
  • Ran gel on PCR from 8th September
    • 150V, 50 minutes
    • No sign of DNA
  • Ladder from Courtney


10th September

  • Ran gel again
  • Split culture
    • 150V, 50 minutes
    • 0.75% gel
    • Ladder from Courtney


11th September

  • FILL IN PCR TABLE


12th September

  • Isolated DNA from 8 cultures
  • Ran gel
    • 1% agarose
    • 150V
    • 38 mins
      • Poor results


15th September

  • PCR PGK Promotor
    • Finnzymes Phusion High Fidelity DNA Polymerase
      • F-530, 20V (2V/uL)
    • FiLL out PCR TABLE


18th September

  • Ran ___? reaction
  • Extracted DNA from gel from 8th September (PGK Terminator)
  • FILL TABLE


19th September

  • Gel of PGK Promotor has no DNA present


23rd September

  • PCR: Fus1 Downstream
  • Fill PCR TABLE


24th September

  • Ran gel of Fus1 Downstream
    • Result: No DNA present on gel


25th September

  • PCR: Fus1 Upstream


1st October

  • PCR: Ste2


8th October

  • PCR: PGK Terminator
    • Use DNA extracted from gel on 18th September
  • Also extracted DNA from gel from 30th September


9th October

  • PCR: Ste2
  • Template used is product from 1st October
  • FIll IN TABLE


  • PCR: Fus1 Upstream
  • Template used was extracted from 30th September on 8th September
  • Fill in PCR TABLE


  • Ran Ste2 gel


14th October

  • Fus1 PCR
  • Template is reaction from 12th October

15th October

  • Gel of Fus1 -> 3 streaks -> no ladder



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