Editing Hot Start PCR using RecA

== Purification ==

Clone into pET vector.

Transform Escherichia coli BL21(DE3)/pLysN&nbsp;cells with plasmid

Grow cells to OD600 &nbsp;0.5 and induced tith 1 mM &nbsp;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)&nbsp;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 &nbsp;-20 oC until use.&nbsp;

== PCR ==

Add TthRecA protein 0.4 ug per 25 ul reaction mixture. The PCR mxi should also contain&nbsp;300 uM ATP.