12 replies to this topic

[aces47]

Hey y'all,
When our lab group went to look at our results for our gel electrophoresis all we could see was the ladders. Can someone please help me in determining why it is that this occurred? I attached a picture of our gel.
Thank You so much!

Attached Thumbnails

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[hobglobin]

Everything might be possible, if you don't give any further description: the PCR or DNA isolation failed, or you forgot to pipet the samples????

[aces47]

hobglobin, on 26 November 2010 - 09:19 AM, said:

Everything might be possible, if you don't give any further description: the PCR or DNA isolation failed, or you forgot to pipet the samples????

Thanks so much! We definitely did pipet the samples so it must be that the PCR or DNA isolation failed.
Do you know what could have caused that?

[hobglobin]

But you know what experiment you did before?  

[aces47]

What do you mean? We took cheek cells and extracted the DNA and ran it through a agarose gel electrophoresis, and for some reason the ladders appeared but the wells containing the cheek DNA did not show up

[hobglobin]

aces47, on 26 November 2010 - 12:45 PM, said:

What do you mean? We took cheek cells and extracted the DNA and ran it through a agarose gel electrophoresis, and for some reason the ladders appeared but the wells containing the cheek DNA did not show up

How should we know this if you don't write it? And what extraction protocol?

[HomeBrew]

If you look closely at some of the center lanes, there is definitely some DNA there.  The rest of the lanes likely have some DNA too, just at a quantity below the detection level of ethidium bromide.  This would make me suspect that your troubles arose from there being very little DNA in the samples you loaded.  Now, the fact that there's very little DNA in your samples could arise from several reasons -- too small a starting sample of cheek cells, loss of DNA somewhere along the recovery procedure, too little an amount of sample loaded on the gel, etc.

[aces47]

HomeBrew, on 26 November 2010 - 01:07 PM, said:

If you look closely at some of the center lanes, there is definitely some DNA there.  The rest of the lanes likely have some DNA too, just at a quantity below the detection level of ethidium bromide.  This would make me suspect that your troubles arose from there being very little DNA in the samples you loaded.  Now, the fact that there's very little DNA in your samples could arise from several reasons -- too small a starting sample of cheek cells, loss of DNA somewhere along the recovery procedure, too little an amount of sample loaded on the gel, etc.

Thank you so much for your help!

[HomeBrew]

You can even go a little further, if you want (this sounds like a school experiment, so I'm going to assume you're a student -- no offense meant if you're not...).  The relationship between the brightness of ethidium bromide stained DNA is directly proportional to the amount of DNA present.  So, if you look at lane seven, the smear you can see in the lower part of the gel (smaller fragments) is about 1/4 - 1/2 as bright as the ladder in that region.  So, if you know the amount of DNA that's present in the ladder in that region, you can estimate how much DNA there was in lane seven for fragments of that size.

[aces47]

HomeBrew, on 26 November 2010 - 06:50 PM, said:

You can even go a little further, if you want (this sounds like a school experiment, so I'm going to assume you're a student -- no offense meant if you're not...).  The relationship between the brightness of ethidium bromide stained DNA is directly proportional to the amount of DNA present.  So, if you look at lane seven, the smear you can see in the lower part of the gel (smaller fragments) is about 1/4 - 1/2 as bright as the ladder in that region.  So, if you know the amount of DNA that's present in the ladder in that region, you can estimate how much DNA there was in lane seven for fragments of that size.

To do this, would it simply be a ratio? I am confused as to how to figure this out because I'm not sure what to compare to. The bands that appeared were from the ladder which obviously don't contain DNA but the same amount of ladder and sample were loaded into the wells. How would we go about the calculations?

[aces47]

HomeBrew, on 26 November 2010 - 06:50 PM, said:

You can even go a little further, if you want (this sounds like a school experiment, so I'm going to assume you're a student -- no offense meant if you're not...).  The relationship between the brightness of ethidium bromide stained DNA is directly proportional to the amount of DNA present.  So, if you look at lane seven, the smear you can see in the lower part of the gel (smaller fragments) is about 1/4 - 1/2 as bright as the ladder in that region.  So, if you know the amount of DNA that's present in the ladder in that region, you can estimate how much DNA there was in lane seven for fragments of that size.

To do this, would it simply be a ratio? I am confused as to how to figure this out because I'm not sure what to compare to. The bands that appeared were from the ladder which obviously don't contain DNA but the same amount of ladder and sample were loaded into the wells. How would we go about the calculations?

[bob1]

The ladder is composed of DNA. Some types of ladder have specifications on how much DNA is contained in each band, so you can do a rough quantitation off the relative brightness of the bands.  Ask your lab demonstrators which brand and the name of the DNA ladder (my guess is 100 bp marker from Invitrogen), then you can look up the ladder on the companies' website and see if there are quantitative bands.

[HomeBrew]

The ladder *is* DNA.  It was purchased from some company at a certain concentration.  Let's say I was using 1 Kb Plus DNA Ladder from Invitrogen (see here).  This ladder is sold as 250 μg at a concentration of 1 μg/μl.

Let's say I loaded 10 μl of this ladder on my gel -- if so, then I've loaded 10 μg of DNA in that lane (waaayyy too much, but let's just continue).

Invitrogen's literature on this ladder says that "the 1650-bp band contains approximately 8% of the mass applied to the gel".  If so, then the 1650-bp band contains 0.8 μg of DNA (or ~0.74 picomoles of DNA for a 1650-bp piece).

If I look at the brightness of the 1650-bp band in the ladder, and compare it to a band on my gel, and it's about the same, then my sample band is present at ~0.74 picomoles; if it's twice as bright, then there's about 1.48 picomoles, etc.