55 C for 15 s, and 72C for 30 s, and a final extension of
72 C for 10 min.
- Prepare a 1% (wt/vol) agarose gel while waiting for the PCR
amplification to complete (seeSubheading2,item 2).
- After PCR amplification, add 10μl6gel-loading dye to the
PCR sample. Load the solution into the solid agarose gel. In a
separate well load an appropriate DNA ladder. Run the gel in a
DNA electrophoresis apparatus at 120 V for 35 min.
- After electrophoresis, view the gel using a UV illuminator.
Correct PCR amplification should lead to a DNA band of
978 bp.
- If the molecular weight of the PCR band is correct, excise the
band containing the amplified gene from the agarose gel by
using a commercial kit for DNA extraction. Elute the DNA in
distilled water and quantify by spectrophotometric analysis at
260 nm wavelength with a quartz cuvette.
- Add the following into the tube containing approximately
500 ng of the purified PCR product purified instep 6:6μl
10 appropriate restriction buffer, 3μl NdeI, 3μl BamHI,
distilled water to 60μl. Mix well and incubate the reaction for
2.5 h at 37C.
- Prepare the pET15b vector (Novagen) as well for cloning (see
Note 1). Add the following into the tube containing approxi-
mately 500 ng of vector: 6μl10appropriate restriction
buffer, 3μl NdeI, 3μl BamHI, distilled water to 60μl. Mix
well and incubate the reaction for 2.5 h at 37C.
- After incubation, add 12μlof6gel-loading dye to both the
gene and the vector tubes. Purify both using a 1% (wt/vol)
agarose gel. Extract the gene and vector from the agarose gel
by using a commercial kit.
- Ligate the gene into the pET15b vector using T4 DNA ligase.
Mix 100 ng of digestedgklgene, 20 ng of digested pET15b
vector, 2μlof10T4 DNA ligase buffer, 1μl T4 DNA ligase,
and distilled water to 20μl. Incubate the reaction for 16 h at
15 C.
- Add the ligation reaction into 50μl of chemically competentE.
coliXL1 Blue cells (Stratagene) (seeNote 2). Incubate the cells
on ice for 30 min. Transform the cells using heat shock treat-
ment. Heat the cells containing the plasmid at 42C for 30 s.
Incubate the cells on ice for 2 min. Add 200μl of LB broth into
the transformed cells. Incubate and shake the cells at 37C for
1 h. Plate 100μl of the transformed cells onto selective LB agar
plates supplemented with 100μg/ml ampicillin. Incubate the
plates at 37C for 16–24 h.
Directed Evolution of Quorum Quenching Enzymes 315
