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Too much failure in electrophoresis, am I doing the gel correctly?


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10 replies to this topic

#1 humalog

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Posted 10 May 2010 - 01:51 PM

Hello.

I do experiments in a lab (I'm still a student) and I always do my agarose gel in a certain way. Most of the times however I don't get the expected bands revealed in the gel after electrophoresis. However I do get all the DNA ladder bands revealed.

I wanted to ask if there is a chance I might be doing something wrong when making up my gel. Or does the fact that the ladder is revealed mean that the gel is OK?

So I will explain how I do the gel for the electrophoresis and please let me know if it is wrong.

1) I add 980ml distilled water with 20ml TE of 50X concentration (I do this much so that I can keep some as stock).
2) I mix and add 100ml of this with 1g agarose powder and microwave this until all powder is dissolved.
3) I add 10ml of cybrsafe or however it is spelled.
4) I mix, pour it in the tray and let it dry for about 30 min.
5) I put the gel in the electrophoresis tank, and then fill the tank with the TE buffer I prepared at step 1.


So does my method seem right to you?
Thanks for listening to me and I look forward to hearing from you.

Edited by humalog, 10 May 2010 - 02:02 PM.


#2 bob1

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Posted 10 May 2010 - 04:01 PM

The gel proceedure seems correct for a 1% gel apart from using TE as a running buffer (should be TBE or TAE or SB) and adding 10 ml sybrsafe, I presume you mean microlitres (ul). The gel should be cool to the touch before you put it in the tank and remove the comb.

If you can see the ladder has separated nicely, then your sample should also have run well. You may need to add more DNA to see bands properly, especially if you are doing a restriction digest.

#3 Lapsang

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Posted 11 May 2010 - 04:21 AM

What do you mean when you say you don't get the expected bands? You see no bands at all or you see bands of unexpected sizes?

In addition to what bob1 already said, you may want to check the concentration of the samples if you haven't already - just to make sure you actually have something there and haven't been really inefficient at some earlier stage of your experiments.

Edited by Lapsang, 11 May 2010 - 04:25 AM.


#4 humalog

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Posted 11 May 2010 - 09:11 AM

Hi

Thanks for your responses.

Yes actually when I was saying "TE buffer" I meant to say "TAE buffer" and when I said "10ml of sybrsafe" I meant "10ul".

So you're right, I must be doing something wrong in my experiments and thanks for your suggestions I'll try adding more of DNA in each well next time.

#5 humalog

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Posted 13 May 2010 - 12:12 PM

Hi

I tried again and it still didn't work. This time I saw no bands at all (but still the ladder was visible clearly).

I have a new suspicion that I am doing something wrong after the PCR (or after the restriction digest- I use both these techniques with no good results).

Before I add the DNA PCR product (or the digested DNA in the case of r. digestion) I always add about 2ul of blue/orange dye 6x from Promega. Then I load samples in gel.

Today I saw that even though the bands were visible in the gel with naked eye (as orange or blue), when I put the gel under UV camera, the sample bands were not visible (only the ladder was).

So I wanted to ask you guys whether this blue/orange dye 6x is supposed to be used just for coloring the bands for observing during electrophoresis, or should it also make the bands visible under UV.

In case the first possibility is correct, then I guess I should also be adding a separate reagent to make the bands visible under UV. What would this be?

Edited by humalog, 13 May 2010 - 12:13 PM.


#6 hobglobin

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Posted 13 May 2010 - 12:31 PM

Hi

I tried again and it still didn't work. This time I saw no bands at all (but still the ladder was visible clearly).

I have a new suspicion that I am doing something wrong after the PCR (or after the restriction digest- I use both these techniques with no good results).

Before I add the DNA PCR product (or the digested DNA in the case of r. digestion) I always add about 2ul of blue/orange dye 6x from Promega. Then I load samples in gel.

Today I saw that even though the bands were visible in the gel with naked eye (as orange or blue), when I put the gel under UV camera, the sample bands were not visible (only the ladder was).

So I wanted to ask you guys whether this blue/orange dye 6x is supposed to be used just for coloring the bands for observing during electrophoresis, or should it also make the bands visible under UV.

In case the first possibility is correct, then I guess I should also be adding a separate reagent to make the bands visible under UV. What would this be?

if you see the size ladder then the gel has enough dye (your sybrsafe) for the DNA, too. The orange and blue bands are the "stop solutions" (marker dye in the loading buffer) visible at normal light to see when the electrophoresis is progressed far enough, i.e. finished....normally when those bands haven't reached the other end of the gel, but depending on your needs about the half of the gel...
Are you sure that the PCR is working?

Edited by hobglobin, 13 May 2010 - 12:35 PM.

One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.

#7 humalog

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Posted 13 May 2010 - 12:40 PM

To clarify, the DNA ladder readily has its own dye (from the moment of buying it). I don't add any dye to it.

I understood what the "stop solution" is, but I want to ask if this solution should also be visible under UV.

My stopping solution is this: http://www.promega.c...productleaf_602

Do you think it should also make the samples visible under UV?
(maybe it's a stupid question but I am relatively a beginner)

#8 hobglobin

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Posted 13 May 2010 - 12:48 PM

To clarify, the DNA ladder readily has its own dye (from the moment of buying it). I don't add any dye to it.

I understood what the "stop solution" is, but I want to ask if this solution should also be visible under UV.

My stopping solution is this: http://www.promega.c...productleaf_602

Do you think it should also make the samples visible under UV?
(maybe it's a stupid question but I am relatively a beginner)

but it has only it's own loading dye too (Orange G, Bromphenol Blue and xylene cyanol) ...you wrote that you add sybrsafe to the gel, that should visualise the DNA and visualises the ladder DNA that you see...the loading dyes you only see as shadows under UV, or not at all, as they won't be fluorescent under UV...
One must presume that long and short arguments contribute to the same end. - Epicurus
...except casandra's that belong to the funniest, most interesting and imaginative (or over-imaginative?) ones, I suppose.

#9 humalog

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Posted 13 May 2010 - 02:03 PM

OK I think I understand now. The sybrsafe is what makes the DNA visible under UV. And the loading dye is just for making it visible under naked eye.

Thanks very much.
:P

Of course this means I'm doing something wrong with my PCR but I'll have to research it. Thanks again.

#10 HomeBrew

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Posted 13 May 2010 - 04:00 PM

Just to be clear: the loading dye is just to monitor the progress of the run -- it does not stain the DNA, and has no role in making DNA visible under any lighting conditions.

#11 humalog

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Posted 19 May 2010 - 08:56 AM

Homebrew, thanks a lot.
Now I definitely got it.




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