Difference between revisions of "Team:Glasgow/Project/Overview/Protocols"

E. coli strains

TOP10:
• F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 ΔlacX74 nupG recA1 araD139 Δ(ara-leu)7697 galE15 galK16 rpsL(StrR) endA1 λ-
• Used for cloning of unmethylated DNA (such as from the distribution plate, or DNA synthesised by IDT)

DH5α:
• F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG Φ80dlacZΔM15 Δ(lacZYA-argF)U169, hsdR17(rK- mK+), λ–
• Used for assembly of plasmids, and to methylate DNA from TOP10 in order to transform into DS941

DS941:
• an MG1655 derivative
• AB1157, recF, lacZM15, lacIq
• Used as chassis

DS941.Z1:
• an MG1655 derivative
• AB1157, recF, lacZM15, lacIq
• extra copy of TetR and LacI
• Used for measurements when extra Lac repressor was required

ER2925:
• ara-14 leuB6 fhuA31 lacY1 tsx78 glnV44 galK2 galT22 mcrA dcm-6 hisG4 rfbD1 R(zgb210::Tn10)TetS endA1 rpsL136 dam13::Tn9 xylA-5 mtl-1 thi-1 mcrB1 hsdR2
• Used when dam methylation was not required

Preparation of CaCl₂ Competent Cells

1. Dilute 400μl of overnight liquid culture into 20ml of broth with any necessary antibiotics to select for any plasmids already transformed into the cells
2. Incubate at 37⁰C, shaking at 225rpm for 90 minutes
3. Spin down for 2 minutes at 7000rpm at 4⁰C
4. Discard supernatant, resuspend pellet in 10ml of 50 mM CaCl2, keep on ice
5. Repeat centrifugation for 2 minutes at 7000rpm at 4⁰C
6. Discard supernatant and the resuspend pellet in 1ml of 50 mM CaCl₂, keep on ice
7. CaCl2 competent cells can be kept on ice in the fridge overnight

Transformation of CaCl₂ Competent Cells

1. 1μl of plasmid DNA was added to 100μl of competent cells
2. Samples were incubated on ice for 20 minutes
3. Heat shock was cried out at 37⁰C for 5 minutes
4. Cells were kept on ice for 2 minutes
5. 200μl of broth was added and the cells incubated at 37⁰C for expression step (time varies dependent on antibiotic resistance gene in the plasmid that has just been transformed into the cells):
• Chloramphenicol resistance = 90 minutes
• Kanamycin resistance = 60 minutes
• Ampicillin resistance = 30 minutes
6. 100-200μl of transformed cells was spread on dried L-agar plates with required antibiotic(s) to select for plasmid(s)
7. Plates were incubated at 37°C overnight

Preparation of Electrocompetent Cells

1. Inoculate 400ml L-broth with 4ml culture
2. Incubate at 37⁰C, shaking at 250rpm until mid-log phase (OD600=0.5-0.7)
3. Split culture into 2 x 200ml samples
4. Chill on ice for 20mins
• Spin 4000 x g for 15minutes at 40C
5. Re-suspend each in 200ml ice cold 10% glycerol
• Spin 4000 x g for 15minutes at 40C
6. Re-suspend each in 100ml ice cold 10% glycerol
• Spin 4000 x g for 15minutes at 40C
7. Re-suspend each in 10ml ice cold 10% glycerol
• Spin 4000 x g for 15minutes at 40C
8. Re-suspend each in 500l ice cold 10% glycerol
9. Aliquot 60l into small eppendorf tubes and store at -700C

Transformation of Electrocompetent Cells

1. Add 1μl of plasmid DNA to 30μl of competent cells
2. Leave on ice for 20 minutes
3. Transfer to pre-cooled electroporation cuvette
4. An electrical pulse was delivered by a Biorad Micropulser and 1ml L-broth immediately added
5. Culture was then transferred to a 2ml nunc tube and incubated at 37⁰C for expression step (time varies dependent on antibiotic resistance gene in the plasmid that has just been transformed into the cells):
• Chloramphenicol resistance = 90 minutes
• Kanamycin resistance = 60 minutes
• Ampicillin resistance = 30 minutes
6. Spread 100-200μl of transformed cells on dried L-agar plates with required antibiotic(s) to select for plasmid(s)
7. Incubate plates at 37°C overnight