Team:Illinois/Bimolecular Fluorescence Biosensor Notebook

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Contents

1st July

  • TE Buffer Recipe
    • Uses
      • TE used to bring up oligos into solution
    • People who know how to do this
      • Joleen, Luke
    • Concentration
      • 10mM Tris
      • 1mM EDTA
      • pH 8.0
    • Method
      • For 500mL volume
        • Add: 0.1861g EDTA; 0.6057g Tris to 1 liter flask
        • Bring it up to 500mL with Deionized water (filtered in ---? container)
        • Put on "corning" mixer; get a larger stir bar from drawer and put on slow ~20 RPM rotation; no heat for 2-3 minutes until fully dissolved.
        • Standardize the pH meter (instructions on sign at prep bench)
        • Put gloves on; put pH electrode in flask but first add stir bar and put on low speed mixing
        • Bring pH to 8.0 (i.e. 8.00 +/- 0.05) by adding base (NaOH) or acid (HCl) in very small drops using a plastic pipette. (Wait for pH meter to eqiullibrate after each drop)

IMP: Put cap back on bottom of pH probe

        • Put coloured tape along side of flask and label with pH, name and iGEM
        • Cover it with heavy duty aluminium foil (just like a square inch to cover the top) and put a strip of autoclave tape along top of foil
        • Optional: out in big plastic autoclave bin
        • Bring to autoclave room; do not use the big "Beta Star" 
    Set on: 15 minutes; ~250 degrees Farenheit = ~121 degree Celcius; "liquid" run; for "operator #" just press enter; then "Run"; tubes = ~ 45 minutes; total to run since it must cool down and decompress
        • (Some extra side notes still to add)
  • TAE Buffer Recipe
  • TBE Buffer Recipe
      • 1 liter of 5x TBE Running Buffer, pH 8.13-8.23
    • Materials
      • Tris-base - 54.0g
      • Boric Acid - 27.5g
      • EDTA - 2.92g
      • DI Water - 1.0L
    • Method
      • Stirred with stirring rod for 3-5 minutes
  • Agarose Gel

2nd July

  • DNA Sequencing - "Core Sequencing " --?
    • (https://unicorm.biotec.uiuc.edu) -> "create login account" -> go to "payment manager"
    • Primer: One end primer, included in per tube, 10uM concentration of primer
    • 30-40 ng/uL? sample concentration
    • Protocols/prep online
    • ---? to view chromatograms on site
    • $5 for sequencing; $2.50 for "--? to load"
    • Purify with PAGE before bringing in

1st July

  • PCR Synthesis of BiFC Genes
    • from "PCR-based Synthesis of Long DNA Sequences" 2006 Nature Protocols
    • Attempted synthesis of Venus Fluorescent Protein halves (N and C term) fused to HIV gp41 epitope
    • Formed gene from ~50nt segment oligos at 30uM
    • PCR reactions as described in paper; 50uL reaction volume
    • 'FivePrime MasterMix' PCR mix
    • PCR protocol as in paper for both N terminal

2nd July

  • Gel #1: Results from July 1 PCR
    • Each lane has 2uL orange/blue loading dye; 1% agarose gel
    • Lane 1: Hyperladder II; 2: 8uL C term whole rxn; 3: Cterm w/o leftmost oligo; 4: C w/o rightmost; 5: Just outer oligos 6-9: Same except N terminal fragments
    • 60 minutes at 140 V then 10 min in EtBr dye
    • No great product

3rd July

  • Gel #2: again of July 1 PCR
    • all lanes have 2uL Loading dye; 1% agarose gel
    • Lanes 1+10=ladder; 2+4=8uL whole N term gene; 6+8=8uL C term gene
    • again, very fuzzy, impure product

8th July

  • Agarose Gel Purification of Oligos
    • Made 4% Agarose gel with 8g Agarose, 40mL TBE, 160mL H2O
    • Made 2% with 4g Agarose
    • Microwave for 1:35 for optional warming
    • Ran total of 2 hours on 80 Volts -> --? (decent?)
  • TAZ side project
    • Adam Z streaked 4 plates with High Elu, Amp m100, Taz Ecoli
    • Check for growth tomorrow
  • Gel Extraction Protocol
    • Use microscope slide to cut gel out
    • Visualize on Bio unit with plastic shield from drawer below unit attached to slide out unit
    • Amount of gel collected(into 1.5mL tubes)
    • Oligo #2: 0.0671g -> 402.6g 
  Oligo #3: 0.062g -> 362.0g
  Oligo #4: 0.1062g -> 637.2g
  Oligo #5: 0.0747g -> 448.2g
  Oligo #6: 0.0585g -> 351.0g
  Oligo #7: 0.0601g -> 360.6g
  Oligo #8: 0.0440g -> 264.0g
  Oligo #9: 0.0482g -> 289.2g

9th July

    • Cells were found in small colonies -> decided to let them grow for 24 more hours
    • Plated 4 more E.coli strains with Xgel (spread)
    • Cultured 4 vials of E.coli with X gel
    • Check back in 36 hours

9th July

  • PCR amplification of July 1 PCR
    • Hope to get more product, specifically C terminal half
    • half of 50uL of pcr reaction volume obtained as 'oligo mix'
    • Tube 1: 25 uL oligo mix +1uL of each outer oligo + 20uL Mango MasterMix
    • Tube 2: 3uL of oligos 1-5, plus 20uL MasterMix; 50uL total volume with added water
    • Tube 3: 3uL oligos 6-10 plus 20uL MasterMix

9th July

  • Gel #4: Analysis of July 9 PCR
    • 1% Agarose gel
    • Lane 1: Hyperladder II; 3: 8uL C-term protein (tube1); 5: 8uL tube2; 7: 8uL tube3
    • Run 50 minutes at 140 Volts
    • Again, lackluster: huge streak seen above and below where we expected product
    • Try Again

11th July

  • Temperature-gradient PCR of Unpurified Oligos
  • Below is the gel of this reaction at different annealing temps
    • 1% agarose gel, run for 50 mins at 140V
    • Lane 1: Hyperladder II; 2: 10uL from rxn with 50° annealing temp; 3: 50.9°; 4: 52.8°; 5: 56°; 6L 58.8°; 7: 59.8; Lane 8: 0.6uL 100bp ladder
    • Some improved results in mid-range of annealing temp, but clearly have grossly impure product and need to regroup
    • Try purifying the 50nt oligos we ordered: likely only ~50% pure from factory

14th July

  • PAGE Purification of Oligos
    • 10% premade PAGE gel w/ 10 50uL wells
    • added 40uL of each oligo to well and 8uL loading dye
    • Just did this for C terminal protein; also only the inner oligos since the outer are smaller and probably already pure
    • Lane 1 and 10 ladder; lanes 2-9 correspond to oligos 2-9 respectively for the C term protein synthesis
    • Loaded into PAGE chamber
    • Ran for 70 mins at 140V
    • Very smeared results-> used too much volume in each well

15th July

  • Redo of PAGE Purification of Oligos
    • Same as July 14th protocol except used 20uL of each oligo instead of 40uL to prevent overflowing
    • Also used 10uL and not 8uL loading dye
    • Ran for 30 minutes at 200 Volts
    • Put in EtBr dye for 15 mins and imaged
    • HUGE streaks for each of our products; either gel was run poorly or we have like 10% purity of oligos
    • Conclusion: this PCR gene synthesis method will not work
    • Review literature for other approaches

20th July

  • Obtained a Citrine Fluorescent protein from UIUC Rao lab
    • On bacterial plasmid
    • Will use instead of synthesizing whole gene de novo
    • Sequence known so can design overlapping primers from our 'novel' part of the gene
    • Instead of fusing HIV gp41 fragment to each FP half, will fuse a poly (6X) His tag
    • Model system to evaluate BiFC assay: His-tag fused to each FP half
    • Bound by mAb against PolyHist -> complementation (hopefully)

30th July

  • NEW Gene Synthesis Protocol
    • From 'Gene2Oligo' tool available online
    • Breaks gene up into ~40nt chunks but OVERLAP FULLY so that even impure oligos can create pure product
    • AminoAcid Sequence: overlap CFP from Rao lab-> 20AA flexible peptide linker-> PolyHis Tag-> stop
    • This ~100 nt sequence will be synthesized de novo w/ Gene2Oligo Method
    • Ordered 6 oligos of ~40nt, brought to 20uM
    • PCR as described by Gene2Oligo paper:
    • 0.1uM final oligo concentration, plus FivePrime MasterMix, plus 25mM MgCl2, plus water to 50uL
    • First performed the Gene2Oligo 'synthesis' step of the protocol

31 July

  • Gene2Oligo 'Amplification' Step
    • As described in paper: 50 uL pcr tube; 20uL MasterMix, 1.5 uL of each 30uM outer primer, 3uL of rxn from July30, plus MgCl2
    • Ran w/ PCR reaction from paper
    • Product added to gel from other IGEM expt: AWESOME, dark, perfect band right where we expect it
    • This method works to synthesize at least smaller (~100nt) segments
    • Also, we have a functional collection of the His-Tag, linker, overlap gene
    • Next step: amplify out the CFP from the plasmid to fuse to this

5th August

  • PCR Amplification of Citrine Fluorescent Protein
    • Ordered primers brought up w/ water to 300uM
    • In each PCR tube: 30uM final concentration of each (forward and reverse) primer, 20uL MasterMix, 1uL out of 50uL plasmid, brought up to 50uL total volume
    • PCR protocol: 90° for 50 sec; 50° for 50 sec; 72° for 1:45; repeat this for 30 cycles, then 72° for 5:00, then hold at 4° for ever

6th August

  • Gel Electrophoresis of 5 August PCR
    • 8uL PCR mixture plus 2uL loading dye
    • 1.5% agarose gel at 200V for about 50 mins
    • EtBr visualization
    • Very dim band where we expect product, also a couple other dim bands
    • Bad gel? Or bad PCR

6th August

  • Revised PCR Protocol for CFP Amplification
    • Same mixture in tube as August 5th
    • BUT: Initial denaturation at 94°C for 2:00
    • then: 94° for 1:00, 47° for 1:00, 72° for 1:00; repeated 25 times; then 72° for 7 mins, then hold at 4°

7th August

  • Gel of August 6 PCR
    • 2% Agarose gel, lane 1= 100bp ladder; land 2= 8uL product + 2uL loading dye
    • EtBr then imaging, a little darker produce but still nothing great, probably not enough to transfect

3rd September

  • PCR Amplification of CFP: Round III
    • Two reactions using the same protocol as on the 6h of august, but with different PCR Mix:
    • 20uL 5Prime MasterMix, OR 25uL MangoMix

7th September

  • Gel of PCR from September 3
    • 1.5% agarose gel, run at 150 Volts for 50 minutes
    • Lane 1: loading dye and 5uL 100bp ladder; 2: 8uL MasterMix reaction; 3: 8uL MangoMix rxn
    • EtBr visualization
    • No great difference between the two pcr mixtures
    • Still no great product

8th September

  • One Last Attempt: PCR Amplification of PCR
    • Increased the concentration of primers: 60uM instead of 30uM
    • Also, used product from the reaction from September 3 instead of raw plasmid (amplify amplification?)
    • Same PCR protocol as on September 3 (except 45° annealing temp)
    • Ran 1% agarose gel for 50 mins at 150V
    • Lane 1: 5uL 100bp ladder; lane 2: 8uL above PCR product
    • Got a decent amount of product, but low purity-> huge smudge
    • Impurity possibly due to re-amplification step
    • Possible villain: low annealing temps
    • Solution: order new amplification oligos with higer temps for annealing

29th September

  • PCR Amplification of CFP Using Longer Primers
    • Longer oligos (~30nt each) used to increase melting temperature; brouth up to 30uM
    • PCR mix: 10uL 2X MasterMix (custom made by Matt); 15uL forward primer; 15uL reverse; 10uL PCR product from Sept 8
    • Same PCR protocol as from September 3, except annealing temp was set to 52° instead of 45

30th September

  • Agarose Gel of Sept 29 PCR
    • 1% Gel run for 50 mins at 150V
    • Lane 1: 5uL Ladder; Lane 2: 8uL Sept 29 reaction product plus loading dye; lane 3 same as lane 2
    • Decent amount of product, but a lot of smearing
    • Possible cause: we again re-amplified an amplification instead of going from original template

30th September

  • PCR Amplification of Original CFP Plasmid with Long Primers
    • Same program as Sept 29 except set annealing temp to 55°C
    • Tube 1: 8uL MasterMix, 15uL forward long primer, 15uL reverse primer, 5uL plasmid, brought up to 50uL total
    • Control tube (2) had Mastermix plus control template and primers.

1st October

  • Agarose Gel of Sept 30 PCR
    • 1% Gel run for 50 mins at 150V
    • Lane 1: 1kbp ladder; 2: 8uL reaction from sept 30; 3: control reaction from sept 30
    • EtBr staining then imaging
    • Way too much ladder (use less in future); also very promising but sort of dark band at ~750nt- Exactly where expected!

2nd October

  • Redo of Oct 1 Gel to Confirm Results
    • Lane 1: 2uL 1kBp ladder; 2: 8uL reaction from sept 30; 3: control from Sept 30
    • Still too much ladder, but otherwise success! Dark band at 750 again
    • Next step-> extract this band

5th October

  • Repeat of PCR Amplification of Original CFP Plasmid with Long Primers
    • Same program and reaction as October 2
    • Then do 1% gel at 150V for 50 mins, EtBr and visualize-> same band
    • Extract this next

7th October

  • DNA Extraction from Oct 5 Gel
    • 'Invitrogen PureLink Quick Gel Kit'
    • Instructions followed directly from kit (see invitrogen website for details)
    • ~150mg isolated from VFP gel around band (with 450uL volume used); also ~140mg from control gel (420uL volume)
    • Next: attempt to quantify DNA
    • [DNA] = 50 ug/ml * Dilution Factor * OD260
    • Tube1 VFP amplification DNA: 0.0055ug/uL; Tube 2 control DNA: 0.186 ug/uL
    • Nice amount of DNA! Next-> clone this into our vector

22nd October

  • Cloning PCR VFP Product into Vector
    • Topo Vector Kit from Invitrogen; kit and protocol available from Invitrogen website
    • VFP solution (VFP1 and VFP2): 1uL salt solution, 1uL TOPO vector; 1uL PCR product from Oct 7; and 3uL H20-> 6uL total volume
    • Control solution (C1 and C2): 1uL salt solution, 1uL TOPO vector; 0.5 uL control PCR product from Oct 7; and 3.5 uL H20-> 6uL total volume
    • Control II (= control 'P') : 1uL pUV19 and One-Shot Cells
    • Plated on LB+AMP
    • VFP1: 200uL transformation mixture; VFP2: 56uL mixture; C1: 200uL; C2: 56uL; P: 10uL reaction mixture plus 20 uL S.O.C.
    • Wait 36 hours for transformation

24th October

  • Results from Oct 22 Transformation Procedure
    • Failure of cells to grow on any of the plates
    • Possible due to insufficient PCR product?

28th October

  • Anoth TOPO Cloning Attempt
    • VFP (from oct 7 extraction): 1uL salt solution; 1uL TOPO vector; 4uL PCR product -> 6uL total
    • Control 1 (C1): 1uL salt; 1uL TOPO; 4uL control PCR product
    • Control II (C2 or CP): 6uL pUC19 plus One-Shot cells
    • Plate on LB + AMP
    • VFP: 200uL; C1: 200uL; C2: 200uL
    • Awaiting Results



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