Could anyone please give me some information about PCR cleanup buffer composition (Prewash buffer, Wash buffer, Binding buffer, Elution buffer) for DNA recovery using silica-based column??
Thanks in advance
Binding buffer: Chaotropic salts that weakens the hydrogen bonding and leads to the DNA binding to silica, with an alcohol added like EtOH or IPA to enhance binding.
like guanidine HCl with IPA (varying)
Prewash: may be lowered amount of chaotropic salts for washing of proteins and pigments.
Wash buffer: for washing of the salts in the sample 100 mM NaCl, 1mM EDTA (pH8.0), 10 mM Tris.Cl (pH 8.0), 50-70% Ethanol
Elution buffer: rehydration of the silica matrix with 10 mM tris HCl pH 8.5 which leads DNA elution. Warmed MilliQ (60 oC) or 1X TE (for long term storage) is also used.
http://bitesizebio.com/13516/how-dna-extraction-rna-miniprep-kits-work/
Binding buffer: Chaotropic salts that weakens the hydrogen bonding and leads to the DNA binding to silica, with an alcohol added like EtOH or IPA to enhance binding.
like guanidine HCl with IPA (varying)
Prewash: may be lowered amount of chaotropic salts for washing of proteins and pigments.
Wash buffer: for washing of the salts in the sample 100 mM NaCl, 1mM EDTA (pH8.0), 10 mM Tris.Cl (pH 8.0), 50-70% Ethanol
Elution buffer: rehydration of the silica matrix with 10 mM tris HCl pH 8.5 which leads DNA elution. Warmed MilliQ (60 oC) or 1X TE (for long term storage) is also used.
http://bitesizebio.com/13516/how-dna-extraction-rna-miniprep-kits-work/
PCR purification kit (Direct purification of PCR products without electrophoresis)
Buffer PB (DNA binding buffer)
5.5 M guanidine hydrochloride (GuHCl)
20mM Tris-HCl pH 6.6 (25℃)
Buffer PE (wash buffer)
20mM NaCl2mM Tris-HCl pH 7.5 (25℃)
80% ethanol
Buffer EB (elution buffer)
10mM Tris-HCl pH 8.5 (25℃)
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