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Tips for visualizing very faint bands in agarose gels? - (The PCR ain't going to get better, so the gel must!) (Apr/28/2010 )

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gebirgsziege on Apr 29 2010, 04:36 AM said:

Together with my previous posters I think you could probably optimise the DNA extraction and therefore reduce background. But it is difficult to detect the DNA of the prey in droppings. How old were your samples when they were collected? I had the experience that the insect DNA degrades rapidly which is probably causing your problems. Bat feces is usually not a too bad template (bad but there are worse things :) to get DNA out from).

You are using a six primer multiplex....would it be possible to "split" into two multiplex PCRs, so you can modify the conditions for the primers better and improve the yield? Or is ther a possibility for a nested approach (like unspecific primers for your gene first for 5 - 15 cycles, cleanup, and then multiplexing)?

For better visualising: loading the whole 20ÁL on the gel might help or longer staining times. Small combs, thin gels, and not too much stain to reduce the background.


Thanks... I do feel like I've spent a lot of time (and even more money) trying to find the best extraction protocol, only to return after 2-3 months of tinkering to what I was using in the first place! My samples are not that fresh (between 2-3 years old), and they were stored in a substandard freezer in a tropical country in a house with regular power outages, so... some of it is just the nature of the beast. I may try a nitrogen tank in the future (another researcher did do this for her plant samples).

I guess I could split up the multiplex, although the six different primers seem to play nice together with regard to to positive control. I think I will try another suggestion of increasing the number of cycles. My concern with a nested approach is that I would have to amplify a fairly large chunk of the COII gene to get all of the primers in there, and I don't want to go much about 500 bp becuse the recovery does drop -- I've had very poor luck trying to amplify anything longer than 700 bp. But it is certainly worth a shot. Thanks!

-KimWG-

Everyone else has really good suggestions for trying to optimise your results, so I can't add anything there.
But I did want to say, DON'T PHOTOSHOP your gel pic if you are going to use it for publication- its just plain dodgy!!

Good luck getting it to work :P

-leelee-

leelee on Apr 29 2010, 07:02 PM said:

Everyone else has really good suggestions for trying to optimise your results, so I can't add anything there.
But I did want to say, DON'T PHOTOSHOP your gel pic if you are going to use it for publication- its just plain dodgy!!

Good luck getting it to work :P


So I can't draw the bands in that I want? :P

-KimWG-

Maybe this is a silly question, but can you add the SYBR to the loading buffer and samples, and then load them on a plain gel? Maybe that could decrease your background and improve visualization of the samples.

edited for grammar.

-lab rat-

KimWG on Apr 30 2010, 12:13 PM said:

leelee on Apr 29 2010, 07:02 PM said:

Everyone else has really good suggestions for trying to optimise your results, so I can't add anything there.
But I did want to say, DON'T PHOTOSHOP your gel pic if you are going to use it for publication- its just plain dodgy!!

Good luck getting it to work :P


So I can't draw the bands in that I want? :P



hahahahaha apparently not- go figure?? :P

-leelee-

Hay
in our lab we're using SYBR safe. Recently a representative adviced GelGreen (from VWR, company in Belgium) or GelRed. I find it much more sensitive then SYBR safe and safer :), doesn't penetrate the skin. Anyhow, our bands were much much brighter
Hopefully useful

-fysio lab-
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