Hot Start PCR using Ecoli SSB
Purification
[edit]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.
PCR
[edit]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