IGEM:MIT/2005/Annie's Notes

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Building/Obtaining Base Parts
(approximate time includes all preparation in the middle such as gather materials and things like that, it's meant to be a guide but they're all dependent on how fast you work)

[[Image:Check.gif]]FecA (3 days) J07017
PCR: primers, E.coli chromosomal DNA, PCR supermix Clean PCR: QIAgen PCR clean up kit Run gel to test PCR: 1% gel, PCR product, 6X sample buffer Digestion on plasmid and PCR product: RE (EcoRI & SpeI), NEB buffer, BSA, water, PCR product Gel purification: PCR product, 6X sample buffer Gel extraction: QIAgen extraction kit, gel Ligation: cut plasmid, insert, ligase buffer, ligase, water Transformation: competent cells (referably chemically competent), ligation product, heat bath/thermocycler, LB, culture tubes, roller drum Plating: plate w/ drug marker, ethanol, spreader, flame ''Approximate time: PCR 2-->3h, clean PCR 15mins, gel 30mins, digestion 2.5h, purification 30mins, extraction 15mins, ligation .5h, transformation & plating 10mins/4plates'' Growing cells overnight in warm room Making overnight culture: LB, culture tubes, flame Miniprep: QIAgen kit, overnight culture, microcentrifuge, cold room Digest for test: RE (EcoRI & SpeI), NEB buffer, BSA, water, miniprep product Run gel: 1% or 2% gel, digest product, 6X sample buffer Freezer stock: 40% glycerol, overnight culture, flame, special freezer tubes Approximate time: growing cells and overnight culture 12-16h, miniprep 2h, digestion 2.5h, run gel 30-45mins, freezer stock 1min/2tubes Notes: EcoRI-NotI-XbaI-FecA-SpeI

[[Image:Pending.gif]]FecA'(3 days) J07018
PCR: primers, FecA, 10X rxn buffer, pWhitescript, control primers 1&2, dNTP mix, PfuTurbo Poly Run gel to test PCR: 1% gel, PCR product, 6X sample buffer Digestion: DpnI, PCR product Transformation: competent cells, polypropylene culture tubes, heat bath/thermocycler, digestion product, LB, shaker Plating: plate w/ drug marker, ethanol, spreader, flame Growing cells overnight in warm room Make overnight culture: LB, single colony, culture tubes Freezer stock: 40% glycerol, overnight culture, special freezer tubes Miniprep: QIAgen kit, overnight culture, microcentrifuge, cold room Sequencing: send to lab Approximate time: PCR 2-->3h, run gel 30-45mins, digestion 1h, transformation & plating 10mins/4plates, grow cells 12-16h, overnight culture 12-16h, freezer stock 1min/2tubes, miniprep 2h, sequencing unkown Notes: EcoRI-NotI-XbaI-FecA-SpeI

[[Image:Check.gif]]FecR (3 days) J07021
PCR: primers, E.coli chromosomal DNA, PCR supermix Clean PCR: QIAgen PCR clean up kit Run gel to test PCR: 1% gel, PCR product, 6X sample buffer Digestion on plasmid and PCR product: RE (EcoRI & SpeI), NEB buffer, BSA, water, PCR product Gel purification: PCR product, 6X sample buffer Gel extraction: QIAgen extraction kit, gel Ligation: cut plasmid, insert, ligase buffer, ligase, water Transformation: competent cells (referably chemically competent), ligation product, heat bath/thermocycler, LB, culture tubes, roller drum Plating: plate w/ drug marker, ethanol, spreader, flame ''Approximate time: PCR 1-->2h, clean PCR 15mins, gel 30mins, digestion 2.5h, purification 30mins, extraction 15mins, ligation .5h, transformation & plating 10mins/4plates'' Growing cells overnight in warm room Making overnight culture: LB, culture tubes, flame Miniprep: QIAgen kit, overnight culture, microcentrifuge, cold room Digest for test: RE (EcoRI & SpeI), NEB buffer, BSA, water, miniprep product Run gel: 1% or 2% gel, digest product, 6X sample buffer Freezer stock: 40% glycerol, overnight culture, flame, special freezer tubes Approximate time: growing cells and overnight culture 12-16h, miniprep 2h, digestion 2.5h, run gel 30-45mins, freezer stock 1min/2tubes Notes: EcoRI-NotI-XbaI-FecR-SpeI

[[Image:Pending.gif]]FecR'(3 days) J07023
PCR: primers, FecR, 10X rxn buffer, pWhitescript, control primers 1&2, dNTP mix, PfuTurbo Poly Run gel to test PCR: 1% gel, PCR product, 6X sample buffer Digestion: DpnI, PCR product Transformation: competent cells, polypropylene culture tubes, heat bath/thermocycler, digestion product, LB, shaker Plating: plate w/ drug marker, ethanol, spreader, flame Growing cells overnight in warm room Make overnight culture: LB, single colony, culture tubes Freezer stock: 40% glycerol, overnight culture, special freezer tubes Miniprep: QIAgen kit, overnight culture, microcentrifuge, cold room Sequencing: send to lab Approximate time: PCR 2-->3h, run gel 30-45mins, digestion 1h, transformation & plating 10mins/4plates, grow cells 12-16h, overnight culture 12-16h, freezer stock 1min/2tubes, miniprep 2h, sequencing unkown Notes: EcoRI-NotI-XbaI-FecR-SpeI

[[Image:Check.gif]]FecI (3 days) J07022
PCR: primers, E.coli chromosomal DNA, PCR supermix Clean PCR: QIAgen PCR clean up kit Run gel to test PCR: 1% gel, PCR product, 6X sample buffer Digestion on plasmid and PCR product: RE (EcoRI & SpeI), NEB buffer, BSA, water, PCR product Gel purification: PCR product, 6X sample buffer Gel extraction: QIAgen extraction kit, gel Ligation: cut plasmid, insert, ligase buffer, ligase, water Transformation: competent cells (referably chemically competent), ligation product, heat bath/thermocycler, LB, culture tubes, roller drum Plating: plate w/ drug marker, ethanol, spreader, flame ''Approximate time: PCR 1h, clean PCR 15mins, gel 30mins, digestion 2.5h, purification 30mins, extraction 15mins, ligation .5h, transformation & plating 10mins/4plates'' Growing cells overnight in warm room Making overnight culture: LB, culture tubes, flame Miniprep: QIAgen kit, overnight culture, microcentrifuge, cold room Digest for test: RE (EcoRI & SpeI), NEB buffer, BSA, water, miniprep product Run gel: 1% or 2% gel, digest product, 6X sample buffer Freezer stock: 40% glycerol, overnight culture, flame, special freezer tubes Approximate time: growing cells and overnight culture 12-16h, miniprep 2h, digestion 2.5h, run gel 30-45mins, freezer stock 1min/2tubes Notes: EcoRI-NotI-XbaI-FecI-SpeI

[[Image:Pending.gif]]scFv+terminal linker (3 days)
PCR#1: primers (linker fwd1 + linker rev1), scFv in plasmid, PCR mix Clean up PCR: QIAgen PCR clean up kit Run gel#1: 2% gel, PCR product, 6X sample buffer, water PCR#2: primers (linker fwd1 + linker rev2), scFv from PCR#1, PCR mix Clean up PCR: QIAgen PCR clean up kit Run gel#2: 2% gel, PCR product, 6X sample buffer, water PCR#3: primers (linker fwd2 + linker rev3), scFv from PCR#2, PCR mix Clean up PCR: QIAgen PCR clean up kit Run gel#3: 2% gel, PCR product, 6X sample buffer, water Store most scFV(PCR#3) in cold room Sequencing: send to Lab Approximate time: PCRs 1h45mins, clean ups 45mins, store 1min, sequencing unkown Digestion on plasmid and PCR product: RE (EcoRI & SpeI), NEB buffer, BSA, water, PCR product Gel purification: PCR product, 6X sample buffer, 1% gel Gel extraction: QIAgen extraction kit, gel Ligation: cut plasmid, insert, ligase buffer, ligase, water Transformation: competent cells (referably chemically competent), ligation product, heat bath/thermocycler, LB, culture tubes, roller drum Plating: plate w/ drug marker, ethanol, spreader, flame Approximate time: digestion 2.5h, purification 30mins, extraction 15mins, ligation .5h, transformation & plating 10mins/4plates Growing cells overnight in warm room Making overnight culture: LB, culture tubes, flame Miniprep: QIAgen kit, overnight culture, microcentrifuge, cold room Digest for test: RE (EcoRI & SpeI), NEB buffer, BSA, water, miniprep product Run gel: 1% gel, digest product, 6X sample buffer Freezer stock: 40% glycerol, overnight culture, flame, special freezer tubes Approximate time: growing cells and overnight culture 12-16h, miniprep 2h, digestion 2.5h, run gel 30-45mins, freezer stock 1min/2tubes Notes: scFv 762bp, scFv(PCR#1) 792bp, scFv(PCR#2) 819bp, scFv(PCR#3) 882bp EcoRI-NotI-XbaI-scFv(PCR#3)-FLAG-SpeI

[[Image:Pending.gif]]FecA-
Make electro competent DY329 cells: lambda red protocol PCR: primers, pSB1AT3-1, PCR mix, thermocycler Clean up PCR: QIAgen PCR clean up kit Transformation: Selection: Approximate time: PCR 2h, purify 45mins, extraction 15mins Notes: Tetracycline Resistance 1.4kb

[[Image:Pending.gif]]Fur-
Make electro competent DY329 cells PCR: primers, pSB1AC3-1, PCR mix, thermocycler Clean up PCR: QIAgen PCR clean up kit Transformation: Selection: Approximate time: PCR 1h, purify 30mins, extraction 15mins Notes: Chloramphenicol Resistance 658bp+promoter

[[Image:Pending.gif]]FecA Promoter (3 days) J07019
PCR: primers, E.coli chromosomal DNA, PCR supermix Clean PCR: QIAgen PCR clean up kit Gel purification: PCR product, 6X sample buffer Gel extraction: QIAgen extraction kit, gel Digestion on plasmid and PCR product: RE (EcoRI & SpeI), NEB buffer, BSA, water, PCR product Ligation: cut plasmid, insert, ligase buffer, ligase, water Transformation: competent cells (referably chemically competent), ligation product, heat bath/thermocycler, LB, culture tubes, roller drum Plating: plate w/ drug marker, ethanol, spreader, flame ''Approximate time: PCR 30mins, clean PCR 15mins, purification 30mins, extraction 15mins, digestion 2.5h, ligation .5h, transformation & plating 10mins/4plates'' Growing cells overnight in warm room Making overnight culture: LB, culture tubes, flame Miniprep: QIAgen kit, overnight culture, microcentrifuge, cold room Digest for test: RE (EcoRI & SpeI), NEB buffer, BSA, water, miniprep product Run gel: 1% or 2% gel, digest product, 6X sample buffer Freezer stock: 40% glycerol, overnight culture, flame, special freezer tubes Approximate time: growing cells and overnight culture 12-16h, miniprep 2h, digestion 2.5h, run gel 30-45mins, freezer stock 1min/2tubes Notes: EcoRI-NotI-XbaI-FecA Promoter-SpeI-NotI-PstI

[[Image:Check.gif]]Promoter X R0011
Grow overnight culture: stab, LB, roller drum Miniprep: QIAgen kit, overnight culture, microcentrifuge, cold room Freezer stock: 40% glycerol, overnight culture, flame, special freezer tubes Approximate time: overnight culture 12-16h, miniprep 2.5h, freezer stock 1min/2tubes

[[Image:Check.gif]]RBS B0030
Grow overnight culture: stab, LB, roller drum Miniprep: QIAgen kit, overnight culture, microcentrifuge, cold room Freezer stock: 40% glycerol, overnight culture, flame, special freezer tubes Approximate time: overnight culture 12-16h, miniprep 2.5h, freezer stock 1min/2tubes

[[Image:Check.gif]]Terminator B0015
Grow overnight culture: stab, LB, roller drum Miniprep: QIAgen kit, overnight culture, microcentrifuge, cold room Freezer stock: 40% glycerol, overnight culture, flame, special freezer tubes Approximate time: overnight culture 12-16h, miniprep 2.5h, freezer stock 1min/2tubes

[[Image:Check.gif]]RBS+GFP+Terminator E0840
Grow overnight culture: stab, LB, roller drum Miniprep: QIAgen kit, overnight culture, microcentrifuge, cold room Freezer stock: 40% glycerol, overnight culture, flame, special freezer tubes Approximate time: overnight culture 12-16h, miniprep 2.5h, freezer stock 1min/2tubes

Assembling of complete system
X+BGS+FecA'-scFv+RBS+FecI+RBS+FecR+Terminator

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