Protein Protocols Protein Protocols

1040 Kipriyanov
2. Run 15–20 polymerase chain reaction (PCR) cycles on a thermocycler. The thermal cycle
is 95°C for 1 min (denaturation), 57°C for 2 min (annealing), and 75°C for 2 min (extension).
At the beginning of the first cycle incubate for 3 min at 95°C and at the end of the
last cycle incubate for 5 min at 75°C.
3. Analyze the amplified DNA fragments by electrophoresis on a 1.5% agarose gel prestained
with ethidium bromide.
3.1.2. Cloning into Expression Vector (see Note 4)
1. Digest 10 µg of appropriate vector with suitable restriction endonucleases in the presence
of alkaline phosphatase (CIP). Incubate at least 2 h at temperature recommended by the
supplier.
2. Purify the PCR fragments and linearized vector by agarose gel electrophoresis followed
by extraction using a QIAquick gel extraction kit.
3. Digest isolated PCR fragments with restriction endonucleases suitable for cloning into the
vector of choice.
4. Remove stuffer fragments and purify the digested PCR products using the QIAquick-spin
PCR purification kit.
5. Ligate the vector and insert using a molar ratio between 11 and 13. The reaction mixture
consists of 50 ng of DNA, 1 U of T4 ligase, ligation buffer, and H 2O to a final volume
of 10–20 µL. Incubate overnight at 16°C.
6. Precipitate the DNA by adding 1/10 volume of 3 M sodium acetate, 20 µg of glycogen,
and 2.5× vol of absolute ethanol. Incubate for at least 3 h at –20°C. Sediment the precipitate
by centrifugation for 15 min at 10,000g (minifuge). Wash the pellet 4× with 500 µL of
80% ethanol followed by centrifugation for 10 min at 10,000g. Allow the pellet to dry at
room temperature. Dissolve the dry pellet in 5 µL of H 2O.
7. Use the products of one ligation reaction for the electroporation of 40 µL electrocompetent
E. coli cells according to the supplier’s protocol. Plate the bacteria on 2YT agar plates
containing 0.1 g/L of ampicillin and 2% (w/v) glucose. Incubate overnight at 37°C.
8. Test individual colonies for the presence of the desired insert by plasmid minipreps (see
Note 5).
3.2. Preparation of Bacterial Culture
1. Inoculate a few milliliters of 2YTGA with an individual bacterial colony and let it grow
overnight at 37°C when using E. coli XL1-Blue or at 26°C when using RV308.
2. Dilute an overnight bacterial culture 40× with fresh 2YTGA and incubate at 37°C
(XL1-Blue) or at 26°C (RV308) with vigorous shaking (180–220 rpm) until OD600 = 0.8–0.9.
3. Harvest bacteria by centrifugation at 1500g for 10 min and 20°C.
4. Resuspend the pelleted bacteria in the same volume of either fresh 2YTSA or YTBS
medium (see Note 6). Add IPTG to a final concentration of 0.2 mM (see Note 7) and
incubate the bacterial culture for 14–16 h with shaking at room temperature (22–24°C).
5. Collect the cells by centrifugation at 6200g for 20 min and either discard the culture
supernatant (RV308) or retain it and keep on ice (XL1-Blue) (see Note 8).
3.3. Isolation of Recombinant Product from Soluble Periplasmic
Fraction and Culture Medium
1. Resuspend the pelleted bacteria in 5% of the initial volume of ice-cold 50 mM Tris-HCl,
20% sucrose, 1 mM EDTA, pH 8.0, and incubate on ice for 1 h with occasional stirring.
2. Centrifuge the cell suspension at 30,000g for 40 min at 4°C, and carefully collect the
supernatant (soluble periplasmic extract). In case of using RV308, go to step 5. If using