The Southern Blot technique

[student12]

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?

[canalon]

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?

[student12]

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?

[canalon]

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

[student12]

Another way of phrasing my previous question is that why do the DNA have to be blot on nitrocellulose/nylon paper?

[canalon]

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.

[kabuto]

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...