Hot Start PCR using Ecoli SSB

Purification

Clone: 2001 pT7A3E-HC-EcoSSB transform into E. coli BL21(DE3) pLysS.

if expression is low, try to clone it into a pET vector with s-tag

At OD600=0.4, Induce with 1 mM IPTG for 5 hours.

Spin down

Freeze

Thaw and Resuspend in 20 ml of buffer A

20 mM Tris, pH 7.9, 500 mM NaCl, 5 mM imidazole, 0.1% Triton X-100

Homogenize, spin

Load supernantant to Ni column

Wash with Buffer A containing 10 mM imidazole

Elute His6-tagged SSB protein with low salt Buffer B

Purify further on QHP.

use 0.5 ug SSB in 50ul PCR mixture

other enhancers:

formamide

dimethyl sulfoxide (DMSO

glycerol

trimethyl ammonium chloride (TMAC)

gene 32 protein from bacteriophage T4

native SSB protein,

Ref