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 (FILL IN PCR TABLE)
tube 1 2 3
Buffer G 12.5uL x4 50uL
template 0.5ul of both heavy and light chains
primers 1ul of appropriate forward and reverse primer
MgCl2 0ul 0ul 3ul 3ul
master mix 20ul
H20 to 50ul total volume

PCR program: 1. 94 degrees 5 min 2. 94 degrees 1 min 3. 50 degrees 1 min 4. 72 degrees 1 min 5. GOTO 2. 29 cycles 6. HOLD at 4 degrees

Products run on a 1.5% agarose gel, no products of 700-800bp.

  • 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

  • FILL IN PCR TABLE


3rd September

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


8th September

  • FILL IN PCR TABLE
  • 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




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