Ulrike Heberlein
PRIMER EXTENSION
All solutions, eppendorfs and pipet tips need to be RNAase-free and sterile.
Use gloves and work carefully (don't sneeze in your tubes).
primer extension;
1) Precipitate the RNA with 5' end-labelled primer in 0.3M NaOAc with 2.5
volumes of ethanol. If working with low amounts of RNA add some yeast RNA
carrier (approx. 10µg). Wash pellet with 70% ethanol and dry.
2) Resuspend RNA in 8 µl ddH2O and add 2 µl of 5X annealing buffer (1.25M KCl,
10mM Tris-HCl pH 7.9, 1mM EDTA). Double this volume if more than 20 µg of
RNA are used, i.e., use 16 µl ddH2O and 4 µl of 5X buffer.
3) Incubate at 60° for 90 minutes. Spin briefly every 30 minutes to avoid drying of
the RNA. The annealing temperature depends on the length and GC content of
the primer. The above is O.K. for 20-30 base primers with 50-70% GC content.
4) Add 23 µl of PE mix (10mM MgCl2, 5mM DTT, 100 µg/ml actinomycin D,
0.33mM dNTP's, 20mM Tris pH 8.7 at room temp., pH 8.3 at 37°C, store at
-20°C in the dark) and 10 U of reverse transcriptase (Life Sciences). Mix
carefuly, spin briefly, mix again. (Use twice these quantities if annealing was
done in 20 µl.)
5) Incubate at 37°C for 1 hour.
6) Add 0.3 ml chilled ethanol. Put in dry ice bath for 15 min. Spin at 4°C for 10
min. Wash in 70% ethanol. Dry.
7) Dissolve in 2 µl of 0.1N NaOH, then add 4 µl of formamide dyes. Boil for 2
min., chill on ice and load on sequencing gel.