Table 1

Summary of HoxD13 primers and PCR experiments

Amplimer

Fingerprint

Primers

Sequences

PCR conditions

size(bp)

Endonuclease

sizes(bp)


95°C 10 min

519

DF1

5'gggagctgggacatgg

Hot Sart,

ac

95°C - 45 sec/

1.1 kb

PpumI

316

64°C - 2 min

40 cycles,

DR2

5'ctggaccacatcagga

72°C 5 min.

265

gaca


Total RNA (3 μg) was denatured together with oligo-dT primer (10 pmol) for 15 min at 68°C. After 5 min incubation on ice, poly-adenosine (poly-A) RNA was reverse-transcribed at 42°C for 90 min in RT solution (50 mM Tris-HCl, pH 8.3; 40 mM potassium chloride; 8 mM magnesium chloride; 0.5 mM each dNTP; 225 ug/ml bovine serum albumin; 5 mM dithiothreitol; 20 units Rnasin and 200 U superscript (Gibco-BRL, Gaithersberg, MD, USA)). The cDNA was incubated at 95°C for 5 min to inactivate the reverse transcriptase, and served as the template for PCR amplification. Sequence information in the genomic clones have been used to generate the PCR primers. The most suitable PCR amplification conditions were determined for each set of primers by on line primer design program (primer3). PCR was performed after adding 80 ul of PCR mixture (50 mM each dNTP; 50 pmol of each sense and the antisense primer; 2.5 units of Taq polymerase (Perkin-Elmer, USA).

Wang et al. BMC Molecular Biology 2000 1:2   doi:10.1186/1471-2199-1-2

Open Data