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yucel 03-02-2007 11:01 PM

no amplification or non specific band
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?

moleculardude 03-06-2007 02:55 AM

Re: no amplification or non specific band
Hello Yucel,
welcome to the forums!
Could you be a bit more specifc. Are the cDNAs all extracted from the same source? If they are not, then it may be expected that some samples will have higher expression than others.

Also if you are preparing the cDNAs, maybe you have prepared some cDNAs different than others. The salt concentration, inhibitory factors or even buffer concentrations may affect the ability of the PCR reaction to occur.

I would suggest checking all your cDNAs first by UV spec (and loading them on a gel) to check quality and quantity.
If they all seem ok, I would only then proceed to PCR. If they are all ok, yet you are still getting problems please post back here.


yucel 03-19-2007 03:47 PM

Re: no amplification or non specific band
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

PeterScherp 03-24-2007 03:54 PM

Re: no amplification or non specific band
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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