Anchoring 3' oligonucleotide primers (300 Mg/ml) in 10 mM Tris-Cl (pH 7.6), 0.1 mM EDTA The anchoring 3' oligonucleotides are a family of 12 primers with the general structure 5'-d(T)12VN-3', where V is either C, A, or G, and N is C, T, A, or G. For example, one primer in the series is 5'-d(T)12CC-3', the next is 5'-d(T)12CT-3', etc.
Arbitrary 5' oligodeoxynucleotide primers (50 Mg/ml) in 10 mM Tris-Cl (pH 7.6), 0.1 mM EDTA Sixteen arbitrary 5' oligonucleotide primers are required, each ten nucleotides in length. The sequence of each primer is chosen at random, but it should contain approximately equal numbers of the four bases, with a balanced distribution of G and C residues, and a low propensity to form stable secondary structures.
Total RNA (100 Mg/ml)
Total RNA extracted from cells with chaotropic agents is generally the template of choice for DD-PCR. RNAs to be compared by DD-PCR should be prepared in an identical fashion. Poly(A)+ RNA is not ideal as a template in differential display.
Was this article helpful?