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[[Image:BiFCnotebookpic.png]] {{bottom_template}} <!--PLACE TEXT BELOW HERE --> == 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 <!-- == Insert Date Here == * lab procedure ** more lab procedure * second lab procedure ** more about second lab procedure COPY/PASTE THEN REMOVE THIS AND BRACKETS--> {{bottom_template}}

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