Reaction conditions.

diff --git a/biblio/8346046.mdwn b/biblio/8346046.mdwn
index c35072543522a5f32d7a830a3fb9218358ce0673..4c42ad74e775b5fadba46587bb060efcf1bc40a7 100644 (file)
--- a/biblio/8346046.mdwn
+++ b/biblio/8346046.mdwn
@@ -7,4 +7,8 @@ Hirzmann J, Luo D, Hahnen J, Hobom G.
 
 Determination of messenger RNA 5'-ends by reverse transcription of the cap structure.
 
-[[!pmid 8346046 desc="The mRNA cap can template an extra C in the first strand cDNA."]]
+[[!pmid 8346046 desc="The mRNA cap can template an extra C in the first strand cDNA. “50% of the cDNAs of capped mRNA molecules had an extra G”"]]
+
+“poly(A)+ RNA was obtained using Hybond-mAP paper (Amersham). 1 µg of poly(A)+ -RNA in 9.65 µL of water was heated to 60°C for 3 min, cooled on ice, added to 4 µL of 5 × RT buffer (1 × RT buffer is 40 mM Tris-HCl, pH 8.3, 5 mM MgCl2, 40 mM KCl, 2 mM DTE), 3 µL dNTPs (10 mM each), 0.25 µL (10 units) of RNasin (Promega), 2.5 µL of XhoI-(dT)17 oligonucleotide primer (300 pmol, 0.5 µg/µL). and 0.6 µL (16 units) of avian
+myeloblastosis virus (AMV) reverse transcriptase (Boehringer).  The reaction was continued at 42 °C for 2 hours, and excess XhoI-(dT)17 primer and substrates were removed by glass powder purification followed by ethanol precipitation.”
+