6 replies to this topic

[josse]

Dear all,

I have the following question: I received a protocol both blottings methodes.
And for the northern blotting one step is to use formamide to prevent or reduce intermolecular basepairing.
Now I am wondering: why do I need to do this only when using northern blotting? And not with southern blotting? Is this because DNA has less intermolecular basepairing?

Or is this just an error in 1 protocol and should I do it in both protocols? Or is it ok just to do the formamide step only when working with RNA (northern) and not when working with DNA (southern)?

thanks

[bob1]

No, it's because DNA is double stranded so it forms linear molecules, whereas RNA is single stranded and can form secondary structures with itself or other strands.  The formamide denatures the RNA keeping it single stranded.

[josse]

Oh I see, thanks a lot

[Feelcontraire]

josse, on Nov 14 2009, 07:33 AM, said:

Dear all,

I have the following question: I received a protocol both blottings methodes.
And for the northern blotting one step is to use formamide to prevent or reduce intermolecular basepairing.
Now I am wondering: why do I need to do this only when using northern blotting? And not with southern blotting? Is this because DNA has less intermolecular basepairing?

Or is this just an error in 1 protocol and should I do it in both protocols? Or is it ok just to do the formamide step only when working with RNA (northern) and not when working with DNA (southern)?

thanks

If you use formamide for DNA you may disrupt the double branch structure.

[josse]

Feelcontraire, on Nov 18 2009, 12:03 PM, said:

josse, on Nov 14 2009, 07:33 AM, said:

Dear all,

I have the following question: I received a protocol both blottings methodes.
And for the northern blotting one step is to use formamide to prevent or reduce intermolecular basepairing.
Now I am wondering: why do I need to do this only when using northern blotting? And not with southern blotting? Is this because DNA has less intermolecular basepairing?

Or is this just an error in 1 protocol and should I do it in both protocols? Or is it ok just to do the formamide step only when working with RNA (northern) and not when working with DNA (southern)?

thanks

If you use formamide for DNA you may disrupt the double branch structure.

Ah I see, thats a second reason then why you can not use formamide for DNA.
In fact you do not need formamide + if you use it: it will be bad for the DNA.

Ok thanks a lot

[josse]

Feelcontraire, on Nov 18 2009, 12:03 PM, said:

If you use formamide for DNA you may disrupt the double branch structure.

And

Quote

No, it's because DNA is double stranded so it forms linear molecules, whereas RNA is single stranded and can form secondary structures with itself or other strands. The formamide denatures the RNA keeping it single stranded.

After rethinking about this, I have however the following remark, question:

I can understand you use formamide to prevent intramolecular binding in the RNA.
However when working with DNA: you will denature this to blot it, so who cant you use formamide then ?
In the end you would need to denature your DNA anyway?

Or does the formamide do more then denature the dsDNA ?

[bob1]

The DNA is run as a native strand, it doesn't really need to be denatured until you are detecting it^*, especially as you are looking for the correct size fragment, whereas with RNA you are trying to keep it single stranded (its native state), so that it runs at the correct size.  To this end, you denature the RNA with heat in a formamide solution and then run it on a gel with formamide to keep it denatured.  There are such things as ssDNA Southerns, especially for detecting oligo-nucleotides and the like.

Quote

However when working with DNA: you will denature this to blot it, so who cant you use formamide then ?
In the end you would need to denature your DNA anyway?

With the DNA you can use NaOH to denature it, why play around with a much more dangerous chemical if you don't need to.

Quote

Or does the formamide do more then denature the dsDNA ?

Actually the formamide just lowers the Tm of the DNA by competing for hydrogen bonds on the bases, so you would need to denature the DNA with heat also, as you do to prepare the RNA for running on a northern.


* As the DNA binds very tightly to the membrane, you "can't" denature it once transferred, so it is done in-gel and then transferred. The only reason it is denatured into single stranded DNA is so that it can be detected.