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
    • W303a S. Cerevisiae

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% agarose, 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
    • 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

  • Split culture
  • Ran gel from 8th September again
    • 150V, 50 minutes
    • 0.75% gel
    • Ladder from Courtney

11th 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

12th September

  • Isolated genomic DNA from 8 cultures of W303a yeast cells
    • Protocol from Wiley's Current Protocols in Molecular Biology
    • Labeled templates 1,2,3,4 and 1a,2a,3a,4a


  • Ran gel of PCR from 11th September
    • 1% agarose
    • 150V
    • 38 mins
      • Poor results

15th September

  • PCR PGK Promotor
    • Finnzymes Phusion High Fidelity DNA Polymerase
      • F-530, 20V (2V/uL)
5x Phusion HF Buffer 10uL x3 30uL
10mM dNTPs 1uL x3 3uL
Primer A(Forward) 1uL x3 3uL
Primer B(Reverse) 1uL x3 3uL
Template(1) 10uL x3 30uL
Phusions DNA polymerase 0.5uL x3 1.5uL
H2O 26.5uL x3 79.5uL

18th September

  • Ran reaction mentioned on 15th September
  • Extracted DNA from gel from 8th September (PGK Terminator)
Tube Gel(g)
1 0.332
2 0.278
3 0.307
4 0.349
5 0.385

19th September

  • Gel of PGK Promotor has no DNA present


23rd September

  • PCR: Fus1 Downstream
  • EPICENTRE Bioetechnologies - MasterAmp(TM) Taq DNA Polymerase
  • Per 50uL of reaction,
MasterAmp Taq 10x PCR Buffer 5uL x3 15uL
1mM dNTPs 1uL x3 3uL
Primer 1 1uL x3 3uL
Primer 2 1uL x3 3uL
25mM MgCl2 6.75uL x3 20.25uL
MasterAmp 10x PCR Enhancer 0 or 15uL x3 45uL
Taq DNA Polymerase 0.25uL x3 0.75uL
template 2 20uL x3 60uL
  • PCR Settings:
    • 4mins, 94 degree celcius
    • 30s, 94 degree celcius
    • 30s, 56 degree celcius
    • 1 min, 72 degree celcius -- to step 2 -- 30x
    • 7 min, 72 degree celcius

24th September

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


25th September

  • PCR: Fus1 Upstream
  • same settings as September 23rd
  • Template 3


30th September

  • PCR: Fus1 Upstream
  • same settings as September 23rd
  • Template 4 and 1a

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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