biochemistry3096

the cDNA template was derived by extracting RNA was reverse transcribing it to make cDNA. Are there any other suggestions for amplifying the wrong fragment during PCR apart from the ones suggested above ? I have checked my primers specificity by conducing a blast search which yeilded my desired amplicon as the only hit, so i dont understand why i amplified the wrong cDNA fragment???

Thank you for your suggestions. I have designed my primers based on general primer design laws e.g primers 18-22bp, 50-60% AT pairing, annealing temp of 55 degrees, ect ect. I have used a blast search to see the hits, and my top hit with 100% is the gene i am looking to amplify, however, this did not happen. I have been using gradient PCR from 50-62 degrees to find the optimal temp, but this has still given me the wrong amplicon. I have also tried altering the primer concentration and Mg concentration but with no success. I think the next thing would be to design another set of primers, but its just seems strange as to why these primers are not working.....

Hi, I have recently designed a set of primers to amplify a fragment of a gene present in an cDNA template. I have performed a blast search on my primers and the results have shown a hit with my gene of interest. However, after conducing the PCR and cloning my fragment into a vector, i sent it off for sequencing. The results were surprising. Sequence analysis showed that the amplified fragment was from a completely unrelated gene. I was wondering, bearing in mind my designed primers are specific for my fragment of interest, why I amplified a non-specific fragment. Any suggestions would help. Thank you, Biochemistry3096