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The Southern Blot technique

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

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The Southern Blot technique

Postby student12 » Wed Jul 05, 2006 9:03 pm

In the Southern Blot technique, how can DNA probes locate the fragment of interest because the DNA fragment containg the fragment of interest is double-stranded....so how does the probe hybridise to it?

Also why blot the DNA on nitorcellulose filter paper?
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Postby canalon » Thu Jul 06, 2006 3:33 am

1- the DNA target is denatured and single stranded. (if you have a protocol you will se there is a denaturation step...

2-I ususally do not use nitrocellulose, too fragile, Nylon is better. but all nylon, I choose a positively cahrged memebrane, can you guess why?
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Postby student12 » Mon Jul 10, 2006 10:55 am

Since DNA are negatively charged, having a positively charged nylon would attract the DNA fragments onto the nylon due opposite charges.
Is this correct?

Also why do they put the DNA fragments on nitrocellulose/nylon? Does it relate to DNA probes hybridising to fragments?
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Postby canalon » Mon Jul 10, 2006 12:02 pm

Yes to the first question.

I don't get your question. The membranes are made of a material that can retain the DNA during all the steps required for hybridization. According to some manufacturers nitrocellulose membrane are a wee bit more specific (less background noise ie, non specific hybridization) than nylon, but cannot be stripped and rehybridized later
Last edited by canalon on Thu Aug 03, 2006 11:51 am, edited 1 time in total.
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Postby student12 » Thu Aug 03, 2006 8:47 am

Another way of phrasing my previous question is that why do the DNA have to be blot on nitrocellulose/nylon paper?
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Postby canalon » Thu Aug 03, 2006 11:53 am

Because you need somethin to "carry" the DNA through the experiment, and the gel would not srvive all the operations. Besides in the gel the DNA would diffuse.
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Postby kabuto » Wed Aug 23, 2006 12:22 pm

student12 wrote:Since DNA are negatively charged, having a positively charged nylon would attract the DNA fragments onto the nylon due opposite charges.
Is this correct?

Also why do they put the DNA fragments on nitrocellulose/nylon? Does it relate to DNA probes hybridising to fragments?


i think his question is what is the properties of nylon /nitrocellulose that enable dna transfer...
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