IGEM:MIT/2005/Annie's Notes

[[../Sensor 2/]] [[../Old Notes/]]

Experimental Care

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)

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

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

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

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

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

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

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

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

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

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

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

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

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

FecA N-mer

PDB page