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Southern Blott

Genetics as it applies to evolution, molecular biology, and medical aspects.

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Postby Jinx » Wed Jan 04, 2012 11:20 am

Hi there,
I am a bit stuck and I wonder if anyone can point me in the right direction?

We have done a gel and the DNA fragments where separated. To my understanding we have to measure how far the fragments have travelled. the smaller fragment have travelled futher than the large fragment. So far so good.

How do we measure the distance travelled? From the top of the band or the bottom of the band?

There are more fragment sizes than bands. I assume it is because the fragment size similar and the fragments have accumulated in the same band. How do I record this?

Any help appreciated

Kind regard

Jinx
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Postby JackBean » Wed Jan 04, 2012 11:30 am

You should measure it from middle of the band.
There is infinite number of possible sizes, but you can only those bands, which you see. I don't understand much to your question.
http://www.biolib.cz/en/main/

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Postby Jinx » Wed Jan 04, 2012 12:09 pm

thank you very much for your answer. I am Swedish so I apologise for the gamma and spelling misstake. Your answer helped.

We have been provided with the DNA fragment sizes, but i think what i need to do is set up a calibration curve and then figure out the fragment size from the calibration curve.
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Postby canalon » Wed Jan 04, 2012 2:10 pm

It seems to me that you are talking about a regular gel before the blotting (I see no mention of probes or anything like that).
In any case you want to measure the distance of migration from the bottom of the well to the middle of the band in the middle of each lane. Then you can compare your distance of migration with a set of DNA fragment of known length that was on the same gel (generally referred to as the DNA ladder, fragment size should have been provided by the manufacturer). The ladder allow you to draw a calibration curve where log(DNA size) is linearly correlated with the distance of migration.

And yes multiple fragments of the same size will give only one band. In this case because you know the expected sizes of your fragments, you just record that band X is likely containing fragments Y and Z.
Patrick

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any proof. (Ashley Montague)
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Postby JackBean » Wed Jan 04, 2012 4:15 pm

for determination of DNA fragment sizes you have many available markers
http://www.biolib.cz/en/main/

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