Yicheng Cao, Xuequn Xu
5.0 µl lambda/Hinf I (10ng/ul) 4.0 µl 5X AMV-RT buffer (USB, Cat# 70218) 0.5 µl 32p- dATP 2.5 µl 3dNTP (-dATP) (0.33 mM each) 0.5 µl AMV-Reverse transcriptase (USB, #700412) 7.5 µl ddH2O Total 20 µl.incubate at 42 °C for 15-20 min. Add 20 µl ddH2O and 80 µl formamide mix (USB sequencing reaction stop solution, Cat.#70704). Aliquot and store at -20 °C. Boil 5 min and transfer to ice before use. Load 1 µl per lane.
Make mixture: 10.0 µl 5X AMV-RT buffer 1.0 µl 32p-dATP 1.0 µl 3dNTP (-dATP) 3.3 mM each 0.5 µl AMV-reverse transcriptase 38.0 µl ddH2OTotal 50 µl for 1 gel (21 samples). Add 2 µl labelling mixture to each sample, mix very well and keep at 42 °C for 15 -20 min. Add 4 µl of formamide mix (Stratagene sequencing reaction stop solution). Boil 3-5 min aand keep on ice immediately. Load 1 µl(Qiagen prep) - 2 µl (AutoGen prep)
N M S C S M S S S M S S S M S S S M S S S M S S S M S S S M N NM = Marker S = Sample to be analyzed on the gel C = Internal Control (loaded in a different lane on a different gel) N = any DNA sample (added to prevent lane bending)