
Northern blotting versus southern blotting
#1
Posted 14 November 2009 - 07:33 AM
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
#2
Posted 15 November 2009 - 03:33 PM
#3
Posted 18 November 2009 - 01:50 AM
#4
Posted 18 November 2009 - 03:03 AM
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.
#5
Posted 30 November 2009 - 06:01 AM
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
#6
Posted 08 January 2010 - 10:35 AM
If you use formamide for DNA you may disrupt the double branch structure.
And
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 ?
#7
Posted 10 January 2010 - 03:23 PM
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.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?
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.Or does the formamide do more then denature the dsDNA ?
* 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.