Team:Illinois/Bimolecular Fluorescence Biosensor Notebook

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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