cDNA synthesis or RT-PCR - (Mar/02/2012 )
Hello, I am new in molecular biology especially in RNA work. I would like to ask question about cDNA synthesis. I had isolated my sample RNA from plant by using Trizol reagent. It give 830ug/ml and ratio of A260/280 is 2 and A260/230 is 1.9. Then I treat my sample with DNase I before synthesis to cDNA by using OligodT(18) only. However when I try to check the quality of the cDNA with my primers, there were no band amplified after PCR. My primers were work well with the genomic DNA from the same sample. Do I still need to purify my RNA before cDNA synthesis even the ratio A260/280 and A260/230 were high and good enough just like mention above?or do the DNase treatment inhibit the cDNA synthesis?please help me..I really don’t know what happened to my cDNA..
did you check your RNA on gel and the most important your cDNA?
You said your primers work well with genomic DNA. Are those primers supposed to actually amplify cDNA, that means, are they in exons?
What type of primers are you using???? Primers for cdna are usually different from the ones used to amplify genomic DNA. AsTrof said, are those primers in exonic regions?? Because if theyare in intron it's normal that they do not work in cDNA.