Basic doubt on restriction digestion - (Jun/04/2009 )
Well we were testing the activity of the DNA ligase in our lab when 2 of our PhD scholars had a heated discussion.
we transformed plasmids in two two conditions, one with ligase and one without ligase.
both of them showed colonies.
case 1:
The plate treated with ligase is bound to ligate as the plasmid is circular and the cells will grow even in an antibiotic marker medium.
case 2:
One person says the colonies are due to incomplete digestion of the plasmid and some of the undigested plasmids get transformed as a circular DNA and colonies appear.
though the plasmids which are digested cannot religate as there is no ligase for the phospho- diesterase activity and even if it gets transformed in the bacterial cells, the DNA will be chopped off by exonuclease activity.
the other person says the plasmid will re-ligate inside the bacterial cell after transformation due to the presence of internal DNA ligases and hence colonies appear.
what i feel is plasmid should get re-ligated due to the conformational change (with sticky ends) and/or internal ligases and/or incomplete digestion. I feel there should be one more control experiment done with alkaline phosphatase to assess the DNA ligase activity.
So please clear my doubt that can the plasmid get re-ligated itself without adding DNA ligase? if so, how?
Please send a copy of your answers to arunsekhar1@gmail.com
Arun on Jun 4 2009, 08:30 PM said:
we transformed plasmids in two two conditions, one with ligase and one without ligase.
both of them showed colonies.
case 1:
The plate treated with ligase is bound to ligate as the plasmid is circular and the cells will grow even in an antibiotic marker medium.
case 2:
One person says the colonies are due to incomplete digestion of the plasmid and some of the undigested plasmids get transformed as a circular DNA and colonies appear.
though the plasmids which are digested cannot religate as there is no ligase for the phospho- diesterase activity and even if it gets transformed in the bacterial cells, the DNA will be chopped off by exonuclease activity.
the other person says the plasmid will re-ligate inside the bacterial cell after transformation due to the presence of internal DNA ligases and hence colonies appear.
what i feel is plasmid should get re-ligated due to the conformational change (with sticky ends) and/or internal ligases and/or incomplete digestion. I feel there should be one more control experiment done with alkaline phosphatase to assess the DNA ligase activity.
So please clear my doubt that can the plasmid get re-ligated itself without adding DNA ligase? if so, how?
Please send a copy of your answers to arunsekhar1@gmail.com
Treat DNA ladder with ligase, and see if the ladder shifts upwards.
Linear DNA can transform cells, albeit at very low efficiency.
Arun on Jun 4 2009, 11:30 AM said:
The plate treated with ligase is bound to ligate as the plasmid is circular and the cells will grow even in an antibiotic marker medium.
This case is beyond me. Treating a plate with ligase, like you spread ampicillin on LB plates or what?
One person says the colonies are due to incomplete digestion of the plasmid and some of the undigested plasmids get transformed as a circular DNA and colonies appear.
though the plasmids which are digested cannot religate as there is no ligase for the phospho- diesterase activity and even if it gets transformed in the bacterial cells, the DNA will be chopped off by exonuclease activity.
the other person says the plasmid will re-ligate inside the bacterial cell after transformation due to the presence of internal DNA ligases and hence colonies appear.
You should be gel purifying your digested vector. If you are using 2 restriction enzymes then that the piece within (insertion place) will be removed and that minimize chances of self-ligation.
I would argue in favour of DNA getting chopped off as every cell has got intrinsic mechanism to fight foreign DNA. (Don't ask me why it doesn't act on plasmid, I don't know)
Your argument does make sense, please do the experiment and try it out.
It will not re-ligate in my understanding.
BTW, the argument is interesting and the bugs do all sorts of stuff that we are not aware of it yet. So I won't spend much time on it, unless I will get an article. Just go on with another round of cloning..