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| Hi, I have a problem abouth my pcr amplification. Some samples (cDNA) which are normally working in the different region, are not working in my protocols but the other samples are working in this protocol. When I changed anneling temperature (-1C), I have seen non specific band at this samples. What should I do now? Thanks |
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| Hi, I have cDNAs which are isolated same protocol at the same time. My samples od is 400-500ul. And I have 4 pcr. My first problem; One of my sample is working for examples in 1 pcr not 2 but I know all my pcr protocols are ok. Second problem; At the same protocols some pcr products have nonspesifik band?? But not at the others!! Thans again Yücel |
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| Hi all, I order to figure out what is wrong in your reactions, you need to be a little bit more detailed on 1. What organisms are you extraction from 2. How do you do reverse transcription 3. What primers are u using 4. What are the primer and DNA concentrations 5. What are the PCR conditions In general, it is helpful to measure DNA concentration but that gives you little info about the Quality of your DNA. If the material you are extracting is old, then your DNA might not be the best for amplification. Hence you get some samples with good PCR bands and others just won't work. Also, if your DNA concentration is too high, you will get a smear rather than a nice band. The same is true for the primer concentration (more than one band when using too much) So, in order to really tell what went wrong, we need more info!!! |
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| amplification , band , specific |
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