Protocol

Bacteria Culture

• Strain

DH5a （F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG Φ80dlacZΔM15 Δ(lacZYA-argF)U169, hsdR17(rK- mK+), λ–）use for cloning

ER2566 （F- λ- fhuA2 [lon] ompT lacZ::T7 gene 1 gal sulA11 Δ(mcrC-mrr)114::IS10 R(mcr-73::miniTn10-TetS)2 R(zgb-210::Tn10)(TetS) endA1 [dcm]）use for expressing

• LB Medium 1 liter

-Add the above ingredients to a flask, and then make up to 1 litre with distilled water

• Antibiotics

• Transformation

-Remove competent cells from freezer and allow to thaw

-Add 3-5 μl of DNA to the cells

-Incubate on ice for 30 min

-Heat shock the cells at 37°C for 2 mins

-Add 1000 μl of LB Broth, and incubate in a shaker at 37°C for 1 hour

-Centrifuge for 3 min at 3,000 g, remove and keep 20 μl of the supernatant, and pour away the rest

-Re-suspend the pellet in the 20 μl of supernatant that was removed earlier

-Spread All onto one plate with anti, and then pour the rest onto another. Incubate the plates overnight at 37°C.

DNA

• Standard PCR

• Standard PCR Program

• Error Prone PCR

Use standard PCR program

• Colony PCR

Make up a mixture as standard pcr without template, Pick colonies from your plate, spot onto your reference grid plate and then pipette up and down in the PCR tube, run as standard pcr program.

• CPEC (Circular Polymerase Extension Cloning)

Make up a mixture as standard pcr without template and primer, add 1ug purified DNA fragment, 200ng LINEAR plasmid backbone, run as standard pcr program.

• Overlap Extension PCR Cloning

Make up a mixture as standard pcr without template and primer, add 1ug purified DNA fragment, 200ng plasmid backbone, run as standard pcr program.

Luciferase Assay

-Remove growth medium from cultured cells.

-Re-suspend cell precipitate in 0.85% NaCl.

-Prepare of bacterial cell lysate using Ultrasonication. Sonics ON 3 seconds, OFF 3 seconds, total ultrasonication time 3minutes.

-Mix 50μl of cell lysate with 50μl of Luciferase Assay Reagent(Luciferase Reporter Gene Assay Kit of Beyotime) and measure the light produced with luminometer.