Heat vs pH during plasmid extraction - (May/02/2014 )
Hallo all,
I have read the following question in a textbook I use, but I can not seem to get the correct answer:
Why do we increase the pH to denature the plasmid and chromosomal
DNA during alkaline lysis rather than using high temperatures, which would also denature DNA?
The only thing I can come up with is this:
- using high temperatures would not destroy the cells , you would still need to add the SDS. This seems however a pretty weird reason why not to use the heat method.
- using heat would be more expensive to denature the DNA?
I also looked up a bit more on this boiling method and it seemed a pretty good method to use.. so I am not sure why this textbook seems so negative about this "high temperature method"..
Any insights?
What does it mean by saying 'denature plasmid'? I have been extracting plasmid by concentional and kit methods for years, never heard of denaturing plasmid. Am I missing something?
I think time is also an important factor in plasmid extraction, so using heat would take more time?
Curtis on Fri May 2 19:10:30 2014 said:
What does it mean by saying 'denature plasmid'? I have been extracting plasmid by concentional and kit methods for years, never heard of denaturing plasmid. Am I missing something?
I think time is also an important factor in plasmid extraction, so using heat would take more time?
I am not sure I understand you.
Denaturing the DNA (plasmid and chromosomal) is pretty much the way how you extract plasmids!
This is a basic step in the protocol!
The alkaline pH apparently more readily digests chromosomal DNA than it does plasmid (perhaps due to length and abundance?) so that the plasmid is preferentially extracted. Heat wouldn't do this to the same level. Having said that, boiling preps are fine and I have used them a number of times.
lyok on Fri May 2 20:51:35 2014 said:
lol, I know that, I just didn't know it's called denaturation. Because in biology the word denaturation is used in many other methods too.Curtis on Fri May 2 19:10:30 2014 said:
I am not sure I understand you.What does it mean by saying 'denature plasmid'? I have been extracting plasmid by concentional and kit methods for years, never heard of denaturing plasmid. Am I missing something?
I think time is also an important factor in plasmid extraction, so using heat would take more time?
Denaturing the DNA (plasmid and chromosomal) is pretty much the way how you extract plasmids!
This is a basic step in the protocol!
When closed circular plasmids are denatured, they reanneal very easily, since the two strands are still linked. This is far less true for genomic DNA, and it tends to remain denatured, making it easier to pellet.
Curtis on Sat May 3 13:52:12 2014 said:
lyok on Fri May 2 20:51:35 2014 said:
lol, I know that, I just didn't know it's called denaturation. Because in biology the word denaturation is used in many other methods too.
Curtis on Fri May 2 19:10:30 2014 said:
I am not sure I understand you.What does it mean by saying 'denature plasmid'? I have been extracting plasmid by concentional and kit methods for years, never heard of denaturing plasmid. Am I missing something?
I think time is also an important factor in plasmid extraction, so using heat would take more time?
Denaturing the DNA (plasmid and chromosomal) is pretty much the way how you extract plasmids!
This is a basic step in the protocol!
ah ok.
For what else is it used than? I pretty much only associate it with dna being denatured and thats it.
phage434 on Sat May 3 17:34:22 2014 said:
When closed circular plasmids are denatured, they reanneal very easily, since the two strands are still linked. This is far less true for genomic DNA, and it tends to remain denatured, making it easier to pellet.
I understand this, but why would heat not work ?
Also: Why is the closed circular plasmid still linked when denatured? It can not denature completely because its all tangled up and thus they can not seperate completely? or?
Two examples:
Proteins are denatured e.g. with heat, high/low pH, etc. The most common use for "denatured" I guess.
Denatured ethanol contains e.g. methyl ethyl ketone to make it undrinkable.
Heat works too (see boiling prep).
Under alkaline conditions, the two strands in non-supercoiled DNA (linear fragments of chromosomal DNA, relaxed circular DNA) separate (denature) and are partially removed from solution (I read that the long ss-fragments also bind to other structures and are tangled up within cell debris and thus are pelleted).
But this does not occur with supercoiled forms of plasmid DNA, because the two strands are intertwined and entangled in a way that prevents them from coming apart (the "linked together"). Therefore, supercoiled plasmid remains free in solution.
As hobgoblin said, un-nicked circular plasmids cannot fully separate when denatured, since the helical DNA strands cannot unwind. Gyrase and topoisomerase act in living cells to allow winding and unwinding when necessary.