From: Charles Plessy Date: Thu, 19 Apr 2018 04:39:20 +0000 (+0900) Subject: More details. X-Git-Url: https://source.charles.plessy.org/?a=commitdiff_plain;h=930518553f5b1372d27df7be9e981f3b01588a54;p=source.git More details. --- diff --git a/biblio/10518626.mdwn b/biblio/10518626.mdwn index f69b0d85..7c7c1f60 100644 --- a/biblio/10518626.mdwn +++ b/biblio/10518626.mdwn @@ -1,5 +1,5 @@ [[!meta title="CapSelect: a highly sensitive method for 5' CAP-dependent enrichment of full-length cDNA in PCR-mediated analysis of mRNAs."]] -[[!tag cap method enzyme manganese]] +[[!tag cap method reverse_transcription manganese template_switching]] Nucleic Acids Res. 1999 Nov 1;27(21):e31. @@ -7,4 +7,4 @@ Schmidt WM, Mueller MW. CapSelect: a highly sensitive method for 5' CAP-dependent enrichment of full-length cDNA in PCR-mediated analysis of mRNAs. -[[!pmid 10518626 desc="In presence of the 5' cap and Mn2+, the SuperScriptII enzyme adds 3-4 extra dC residues to the first strand cDNA."]] +[[!pmid 10518626 desc="Extra cytosine are more frequently added in presence of the 5′ cap. Increasing Mg2+ to 6 mM increases the frequency of addition of more than 1 C, but only moderately. Instead, when adding BSA in the reaction, supplementing it with 1 or 2 mM Mn2+ after 1h, 3-4 extra dC residues are added to most of the first strand cDNAs. Standard reaction: 0.75 μM oligo dT (or 0.25 μM gene-specific RT primer); 50 mM Tris-HCl (pH 8.3); 75 mM KCl; 3 mM MgCl2; 5 mM DTT; dNTPs 1mM each; 0.1 mg BSA; 20 U RNAse inhibitor (Roche), 200 U SuperScript II; 1h at 42 °C."]]