https://eln.excellgen.com/index.php?action=history&feed=atom&title=Hot_Start_PCR_using_Ecoli_SSB 2026-10-19T03:25:52Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=Hot_Start_PCR_using_Ecoli_SSB&diff=15587&oldid=prev 2017-02-21T03:07:27Z

<p></p> <p><b>New page</b></p><div>== Purification ==<br /> <br /> Clone: 2001 pT7A3E-HC-EcoSSB transform into E. coli BL21(DE3) pLysS.<br /> <br /> if expression is low, try to clone it into a pET vector with s-tag<br /> <br /> At OD600=0.4, Induce with 1 mM IPTG for 5 hours.<br /> <br /> Spin down<br /> <br /> Freeze<br /> <br /> Thaw and Resuspend in 20 ml of buffer A<br /> <br /> 20 mM Tris, pH 7.9, 500 mM NaCl, 5 mM imidazole, 0.1% Triton X-100<br /> <br /> Homogenize, spin&amp;nbsp;<br /> <br /> Load supernantant to Ni column<br /> <br /> Wash with Buffer A&amp;nbsp;containing 10 mM imidazole<br /> <br /> Elute His6-tagged SSB protein with low salt Buffer B<br /> <br /> Purify further on QHP.<br /> <br /> <br /> <br /> == PCR ==<br /> <br /> use 0.5 ug SSB in 50ul PCR mixture<br /> <br /> <br /> <br /> other enhancers:<br /> <br /> &amp;nbsp;formamide<br /> <br /> dimethyl sulfoxide (DMSO<br /> <br /> glycerol<br /> <br /> trimethyl ammonium chloride (TMAC)<br /> <br /> gene 32 protein from bacteriophage T4<br /> <br /> native SSB protein,<br /> <br /> Ref<br /> <br /> http://www.chimerx.com/store/dna-rna-modifying-enzymes/single-stranded-dna-binding-proteins/single-stranded-dna-binding-protein-e-coli-500ug-436</div>

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