I got troubles that I PCRed certain genes from the Arabidopsis phage cDNA library (from Arabidopsis Biological Resource Center ) these days, but all of the results of PCRs only displayed a few nonspecific amplifications. All of my target genes are 2000bp+, however the nonspecific bands gathered in 200-500bp.
Now, should I make sure of these things at first?
1.The quality of cDNA, because it was kept in -80 for 2 years+ and not used before. Is it necessary to use beta-actin to test the cDNA?
2.The concentration of cDNA template is unknown, so I'm no sure if the content of template I added(2ul cDNA for 50ul) is below the requirement. Could the presence of nonspecific bands revealed the template content is enough in fact?
Need for your suggestion, thanks so much
-Chenjun-
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