Hi everyone, We are conducting gels to determine the structure of fungal community in soils. When we disclose the gels, the bands do not separate and they appear together in the middle or at the end of the gel. The general conditions that we use are acrylamide 8%, 20V-15min and subsequently 70V-840min. With these conditions, bands kept together and do not separate by all the gel. That is why we changed the voltage, running time and even the percentage of acrylamide from 8% to 6%. This last change seems that it has helped us to separate a little more but we do not have the expected result.
We have been doing DGGE for a while (on bacterial, fungal, AMF communities) and this is the first time we experience this problem... does anyone had a similar problem? I upload photos of the gels
I will appreciate any comment or suggestion you can give me.
Thanks a lot!
Hello,
You can visualize clear separation of bands on gel only if the difference between amplicon/DNA sizes are ~5bp. For better separation I would increase the acrylamide concentration from 8 to 10 or 12% and run it for 8 hrs at 150V (or until you see loading dye cross halfwy mark.)
Vijaya
First: do you decide to amplify all ITS or a specific region? In my case I amplified the ITS1 adn the condition of the Gel (80% denaturatiog solution for 8% gel). I prepare a lower solution at 20% and high solution at 50%. I ran the gel in TAE 1% for 16H at 60 V and 60°C.
Please tell us what primers did you used and what was the denaturing gradient ?
Hi Jarosław Grządziel,
We are using the primers ITS4-GC and ITS1F. We started whith the denaturing gradient 20%-70% and then we reduced the gradient to 28%-32%.
Yaiza Lechuga-Lago ,
I would recommend to use different primers, because this pair generates a bit too large product (for the DGGE purpose) ~ 600 - 800 nt, while DGGE works best when using shorter products (200-300 nt).
It's good idea to perform nested-PCR, for example first PCR reaction using your primers, and next using e.g. ITS-1, ITS-2 primers that generate shorter products. Alternatively use just ITS1, ITS2 primers (or another pair generating shorter products) in classic PCR.
This pair works very well with 8% gel, using 20-50% denaturing gradient for ~16h at 60V, 60 C.
Best wishes
Yaiza Lechuga-Lago
We have analysed fungal community structures in the past without any problem. Fingerprinting of fungal community was undertaken in a nested PCR approach using the primers ITS-1F and ITS-4 in the first step. This product was tenfold diluted and used as template in a nested PCR using ITS-1FGC primer with a 40 base GC clamp to the 5' end of the primer and ITS-2 reverse primer. DGGE (10 % gels) was run using a gradient of 20–55% at 63V for 16.5 hours. Check the publications from my group for specific details.
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