7 replies to this topic

[Baconman]

Hi!

I sometimes get a dark zone in the middle of the bands (both ladders and the PCR products) when I run my genotyping PCR on agarose gel. What causes this phenomenon?

Some experimental conditions:
5ul PCR reactions with two sets of primers, the lanes should have up to 4 bands. The PCR buffer is "ready to load"
1.5% agarose gel made with 1x TAE buffer, run at 80V in 1x TAE buffer
I dilute the samples 1:3 with 1x loading buffer and load 1ul of the diluted sample (I often get smeared bands otherwise)
Ladders are 1kb and 50bp ladders

[bob1]

Sounds like an image processing problem - saturation of the image results in "burn out" of the centre of the bands. Shorter exposures or an increase in the f stop/aperture should prevent this.

[Baconman]

It happens also for very weak bands, could it still be saturation?

I tried to attach an image so you can see for yourself, but I am obviously not smart enough to figure out how...

[bob1]

That doesn't sound like saturation. What device are you using for imaging, what dye, and what exposure settings?

 

I think you should be able to attach and image. There's a little icon on the control panel - looks like a square with a smaller green rectangle inside. If that is grey, you might not have enough posts to be able to add images. If that fails, PM it to me and I'll post it.

[Baconman]

Bio-Rad ChemiDoc MP, automatic exposure and GelRed

 

The icon is not grey, but it still does not work for me. Tried sending it to you but that also did not seem to work although it looked like the image was attached

[bob1]

I've been pm'd the image and it is attached below.

 

Having had a look at the image, I think it is probably caused by underfilling of the wells. There are three options - either use a narrower comb so that you get a thinner band, or mix the contents in the well a little bit, or add more sample to each well.

 

It is definitely not a saturation problem - saturation is shown by the red patches on the ladder bands.

Attached Thumbnails

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[Baconman]

Thanks!
I did load a very small volume (1ul) so that might be an issue. I will try to dilute the samples more and load a larger volume.

[mdfenko]

To me, it looks like there may be a “skin” in the wells that are retaining part of the sample, sort of giving 2 origins.

 

you can try flushing the wells to clear any gel from inside or against the glass.

 

also, don’t let the gel sit after loading. Start the current immediately or the sample will start to diffuse into the gel.