DGGE was carried out on 7.5 % polyacrylamide gel in 1× TAE buffer with a denaturing gradient from 30% to 60% (at 60 °C; 70 V; 16 h).
Each lane were loaded with 700 ng of DNA (PCR amplicon of GC968f/1401r).
Attached is the gel image after stained for 30 min with SybrGreen I Nucleic Acid Gel Stain.
Should I adjust the denaturing gradient and reduce the DNA amount?
I'm not a DGGE expert but I have some experience. What is the thing that looks like a ladder in every 5th lane?
Your band doesn't seem to be separating out. I'd try reducing the size of your gradient - maybe try 40 or 45% - 60%? Your DNA has all moved quite far down the gel which would suggest that no denaturing is happening at 30% or even 40% by the looks of it, assuming your gradient is formed properly. You could also try reducing to 50V for 16 hours.
Good luck!
Lindsay
There is a lot of factors that you can vary on the DGGE. All the values that you mention seems right, although perhaps you are using few DNA material, try with more, and of course make sure that you are forming the gradient properly.
Plus, I have the same cuestion that Lindsay: What is the thing that looks like a ladder in every 5th lane?
Clearly you need to improve your denaturating conditions because the total amplicon its looked. Probe with a wide range of concnetrations e.g. 20-80; 30-80, etc.
Hello, Muhammad
I´m not an expert neither, but based on my experience, I recommend you 2 things to do:
* As Dulce Flores wrote, i agree you should try with more DNA. In my first DGGE gels, i loaded few PCR product, and what I did was to amplify 3 or 4 PCR reactions PER SAMPLE, to obtain more DNA. After that, i collected the PCR products in a single microcentrifugue tube and i purified it as a single PCR product. That way, i obtained more PCR product ready to load the gel.
* I also recommend you to purify your PCR products before you load it in the gel, maybe that way the bands look sharper. In my first gels, the bands of my samples also showed smears, but after i purified them with a cartridge kit, the bands were more defined.
Good luck!
Martha
I don't think it is a problem of the gel and gradient. The PCR products might be not very good. I suggest you re-amplify your sample.
I think you have a problem with the gel staining. Stain the gel with silver instead of SybrGreen, silver is way better. I see some big bands in all of the lanes, so I think you have more product but you can not see them, then you will be able to determinate the correct amount of PCR products to be used in your gel. And I know you used some DNA ladder is a good practice to compare the bands.
I had the same problem until some time ago. After many attempts, now I have a good fingerprinting. My main suggestions are:
1) verify the PCR products.
2) change the denaturant gradient as Lindsay Fulton suggested; I have good results with 35%- 50% .
3) change the TAE concentration. I now use 0.5 X
4) reduce the amount of amplicons loaded. I use 100-200 ng/ ul
good luck
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