PRIMER LABELLING FOR PRIMER EXTENSION ASSAY and PRIMER EXTEN

Friday, November 21, 2003

Description
PRIMER LABELLING FOR PRIMER EXTENSION ASSAY and PRIMER EXTENSION

Procedure
Mix in a 20ul final volume:

2ul 10x kinase buffer

1ul oligonucleotide primer (5pmoles/ul)

200uCi 32P-gATP (crude, ICN, 7000Ci/mmole)

10 U polynucleotide kinase

ddH2O to 20ul

Incubate at 37C for 30-45 minutes. Heat at 65C for 15 minutes.

To remove unincorporated label precipitate as follows:

Add 60ul TE + 320ul 2.5M NH4OAc + 20ug RNA carrier + 650ul 100% ethanol (follow this order of addition; don't add the carrier RNA to the reaction first or some RNA will get labelled). Chill on dry ice for 5-10 minutes. Spin in the cold for 15 minutes. Discard supernatant (very hot!!). Resuspend pellet in 180ul TE and add 20ul 3M NaOAc + 500ul 100% ethanol. Chill on dry ice for 15 minutes, spin in the cold for 10 minutes, wash with 70% ethanol, dry and resuspend in 100ul TE (should be ~200cps/ul on minimonitor).

10x kinase buffer:

500mM Tris pH 8.0

100 mM MgCl2

10mM spermidine

1 mM EDTA

50mM DTT (add fresh before using)

All solutions, eppendorfs and pipet tips need to be RNAase-free and sterile. Use gloves and work carefully.

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. 10ug). Wash pellet with 70% ethanol and dry.

2) Resuspend RNA in 8 ul ddH2O and add 2 ul of 5X annealing buffer (1.25M KCl, 10mM Tris-HCl pH 7.9, 1mM EDTA). Double this volume if more than 20 ug of RNA are used, i.e., use 16 ul ddH2O and 4 ul 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 ul of PE mix (10mM MgCl2, 5mM DTT, 100 ug/ml actinomycin D, 0.33mM dNTP's, 20mM Tris pH 8.7 at room temp., pH 8.3 at 37C, store at -20C 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 ul.)

5) Incubate at 37C for 1 hour.

6) Add 0.3 ml chilled ethanol. Put in dry ice bath for 15 min. Spin at 4C for 10 min. Wash in 70% ethanol. Dry.

7) Dissolve in 2 ul of 0.1N NaOH, then add 4 ul of formamide dyes. Boil for 2 min., chill on ice and load on sequencing gel.

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PRIMER LABELLING FOR PRIMER EXTENSION ASSAY and PRIMER EXTEN