Team:Illinois/Bimolecular Fluorescence Biosensor Notebook

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

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
 * 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"


 * (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

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
 * 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

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