Hot Start PCR using RecA
Purification
[edit]Clone into pET vector.
Transform Escherichia coli BL21(DE3)/pLysN cells with plasmid
Grow cells to OD600 0.5 and induced tith 1 mM IPTG for 4 hours.
Spin down cells
Resuspend in 50 mM Tris–HCl (pH 8.0), 5 mM EDTA, 8.4 mM b-mercaptoethanol, 25% (w/v) sucrose and 1 M KCl
Freeze and thaw.
Homogenize.
Heat treatment for 60 min at 65 oC
Spin to remove the precipitate.
Load to Ni column
Elute with Buffer B (low salt).
Dilute (1:1 v/v) with a buffer containing 20 mM potassium phosphate (pH 6.5), 1 mM EDTA, 5 mM b-mercaptoethanol and 10% (v/v) glycerol,
Load to a phosphocellulose (P11 Cellulose; Whatman, Japan) column that had been equilibrated by using the same buffer.
Elute RecA protein with a linear gradient of KCl (0–1.5 M)
SDS-PAGE analysis.
Pool fractions containing the RecA protein
Dialyze against a buffer consisting of 50 mM Tris–HCl (pH 7.5), 0.1 mM EDTA, 5 mM b-mercaptoethanol, 1.5 M KCl and 10% (v/v) glycerol.
Store at -20 oC until use.
PCR
[edit]Add TthRecA protein 0.4 ug per 25 ul reaction mixture. The PCR mxi should also contain 300 uM ATP.