Why I can see non-specific product and I can't see my exprect PCR product. Half a year ago I prepared PCR and this reaction was working in this time. Last time I prepared PCR and my reaction didn't work. I try optymalize my reaction again. I used many of ways. I check my reagents and a lof of stuff. When I prepared my PCR at the other lab (at other department)this reaction was working (I used the same DNA, reagents, water). After I prepared my PCR in my lab and this reaction didn't work (I used the same DNA, reagents, water). In my reaction is a non-specific product that doesn't interfere with reading result. This product also appeared when my reaction was working and appears now but the expected product is missing.
I'll be grateful for help
Hi Adrian,
I would suggest to use the clean environment for your PCR works. Based on your write-up, you don't have a problem with the Primer. However, better to rethink about your primer design and check those are very specific to your gene of interest. Any way always try to avoid the contamination of Pre and Post PCR reactions.
Good luck.
What did change?
Samplers?
PCR machine?
etc
So do quality control.
Good luck
Hi there
Results of PCR just depend on the clear factors like source and purity of your samples, reagents and their quality, accurate protocol, and well-established instruments, you must check each one of these items...
Hope you will do and success.
I don't understand why the previously expected products were visible and non-specific products. Currently, I only see a non-specific product.
Nothing has changed. I do it on the same reagents, the same DNA, primers and thermocycler. It was only when I made PCR in another laboratory (on another thermocycler) that PCR worked. My expected products have appeared.
you said that you used another machine so I think your machine has some problems.
My other PCR reaction also does not work on this machine and it work earlier. I thought it was the reagent's fault, but as I used the same reagents in another laboratory, the reaction worked.
@ Adrian Bakula . When I look the gel picture , it is almost clear you have some contamination problem. Mainly better to change your water first. use the mili-Q water/ nuclease free water. try to avoid contamination by making aliquotes.
I think your machine can't give you right temperature.
Also I think your primer designed in a way that annelling temperature of them is very inflexible therefore you can check it with gradients PCR.
Good luck.
Hi firstly try to clean the working area. seperate the running area than the prepared area. check with the company PCR machine. try toadd in your reaction positive control
I prepared another PCR and this reaction is working in other thermocycler. Ealier the same reaction didn't work in the same therocycler.
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