Paul.xu: Created page with "== Purification == 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..."

2017-02-20T01:07:34Z

Created page with "== Purification == 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..."

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== Purification ==

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 ==

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

Hot Start PCR using RecA - Revision history

Paul.xu: Created page with "== Purification == 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..."