( a probe in other analytical techniques...)RT-PCR Primer Sets for Human and Mouse Mismatch Repair Genes

a probe in other analytical techniques, such as Northern blot hybridization, nuclease protection assays or in situ hybridization. In addition, these probes might be useful for screening cDNA libraries for gene homologues in related species. Because the mismatch repair genes serve an important function in maintaining the genetic integrity of the cell, they are likely to be expressed in detectable levels in most cell types. In this regard, they could be classified as housekeeping genes. Thus, the RT-PCR primer sets could be used as positive controls for cDNA templates in RT-PCR studies unrelated to mismatch repair.

PCR Amplifications Using Primer Sets

figure 1

The human RT-PCR primer sets for mismatch repair were used in amplification reactions containing either cDNA template from the human promyelocytic cell line (HL60) or human genomic DNA template (figure 1). Total RNA from the HL60 cell line was isolated using Stratagenes RNA Isolation Kit and quantitated by measuring absorbance at 260 nm. First-strand cDNA was synthesized in a 50-l volume using 5 g of total RNA and Stratagenes RT-PCR Kit according to the recommended protocol. Either 1 to 2 l of the cDNA or 25 to 50 ng of the human genomic DNA was used in 50-l PCR reactions using RoboCycler temperature cyclers. Each reaction was performed in 1X Taq polymerase buffer, 250 M of each dNTP, 10 pmol of each primer and 2.5 U of Taq2000^TM DNA polymerase. The temperature cycling profile was as follows: One cycle at 95C for 3 minutes; 30 cycles of 95C for 30 seconds, T_A (the recommended annealing temperature for each primer set) for 1 minute and 72C for 1 minute; one cycle at 72C for 10 minutes.
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