Ligation of incompatible overhangs - (Jul/14/2006 )
Hi fellows,
I want to know what happens in a ligation reaction when you've got cohesive overhangs in the 3'end but in the 5' end you've got incompatible overhangs. I know that the 3' end it's going to ligate well, but I'm not sure what is going to happen in the other end.
I want to know what happens in a ligation reaction when you've got cohesive overhangs in the 3'end but in the 5' end you've got incompatible overhangs. I know that the 3' end it's going to ligate well, but I'm not sure what is going to happen in the other end.
In theory the incompatible overhangs shouldn't ligate because the two overhangs are not complementary.
hi
i agree with DNAfactory.
In case of ligation, almost every possibility can happen, ranging (base per base) from reconstituing the 1 site or keeping 5/4/3/2/1 or 0 bases of this site..
In every case, sequencing is the only true method.
But it gonna be a crzay cloning.
I want to know what happens in a ligation reaction when you've got cohesive overhangs in the 3'end but in the 5' end you've got incompatible overhangs. I know that the 3' end it's going to ligate well, but I'm not sure what is going to happen in the other end.
If you're cloning to express protein, you might want to have the 5' end of the construct as the clean and simple one, and let the 3'end work itself out.
One option you could follow is to chase your ligation reaction with some dNTPs and Klenow, just to fill in any overhangs, then transfect into cells...
[/quote]
If you're cloning to express protein, you might want to have the 5' end of the construct as the clean and simple one, and let the 3'end work itself out.
One option you could follow is to chase your ligation reaction with some dNTPs and Klenow, just to fill in any overhangs, then transfect into cells...
[/quote]
No, my sequence isn't for protein expression. I am trying to delete a promoter sequence from its 5' end. So there's no need to be very careful with the 5' end.
You've gave me a great idea: I'm going to perform ligation in presence of klenow!!
I wonder how much well does klenow work in the ligation mixture? Anyway I'm gonna try.
What if do I make ligation, run a gel of it and then purify linealizated plasmid and then I refill with klenow?
Thanks
[quote name='Marvilla' date='Jul 20 2006, 06:42 PM' post='60559']
[/quote]
If you're cloning to express protein, you might want to have the 5' end of the construct as the clean and simple one, and let the 3'end work itself out.
One option you could follow is to chase your ligation reaction with some dNTPs and Klenow, just to fill in any overhangs, then transfect into cells...
[/quote]
No, my sequence isn't for protein expression. I am trying to delete a promoter sequence from its 5' end. So there's no need to be very careful with the 5' end.
You've gave me a great idea: I'm going to perform ligation in presence of klenow!!
I wonder how much well does klenow work in the ligation mixture? Anyway I'm gonna try.
What if do I make ligation, run a gel of it and then purify linealizated plasmid and then I refill with klenow?
Thanks
[/quote]
I would advise you to digest first the extremity you want to fill in, then purify your DNA, then digestion with the other enzyme and finally the ligation. I know that's time consuming but this is the cleanest way of doing it
[quote name='dnafactory' date='Jul 20 2006, 09:53 AM' post='60561']
[quote name='Marvilla' date='Jul 20 2006, 06:42 PM' post='60559']
[/quote]
If you're cloning to express protein, you might want to have the 5' end of the construct as the clean and simple one, and let the 3'end work itself out.
One option you could follow is to chase your ligation reaction with some dNTPs and Klenow, just to fill in any overhangs, then transfect into cells...
[/quote]
No, my sequence isn't for protein expression. I am trying to delete a promoter sequence from its 5' end. So there's no need to be very careful with the 5' end.
You've gave me a great idea: I'm going to perform ligation in presence of klenow!!
I wonder how much well does klenow work in the ligation mixture? Anyway I'm gonna try.
What if do I make ligation, run a gel of it and then purify linealizated plasmid and then I refill with klenow?
Thanks
[/quote]
I would advise you to digest first the extremity you want to fill in, then purify your DNA, then digestion with the other enzyme and finally the ligation. I know that's time consuming but this is the cleanest way of doing it
[/quote]
Oh Thanks! It's the rigth choice!!
I wonder how much well does klenow work in the ligation mixture? Anyway I'm gonna try.
What if do I make ligation, run a gel of it and then purify linealizated plasmid and then I refill with klenow?
Thanks
A word of caution about simultaneous ligation and in-fill. You may well be blunt-ending your cohesive termini (both insert and vector, thus losing your directionality of cloning), rather than ligating in and just tidying up the ends...
I'm also not sure how well Klenow works at ligation reaction temperature.
Also, not sure if this is what DNAfactory meant, so I'll ask the question. Do you mean digest 5' end, fill in, purify, then digest the other end purify and finally ligate? If so, I agree totally, that is the cleanest way to do the job.
You've gave me a great idea: I'm going to perform ligation in presence of klenow!!
I wonder how much well does klenow work in the ligation mixture? Anyway I'm gonna try.
What if do I make ligation, run a gel of it and then purify linealizated plasmid and then I refill with klenow?
Thanks
A word of caution about simultaneous ligation and in-fill. You may well be blunt-ending your cohesive termini (both insert and vector, thus losing your directionality of cloning), rather than ligating in and just tidying up the ends...
I'm also not sure how well Klenow works at ligation reaction temperature.
Also, not sure if this is what DNAfactory meant, so I'll ask the question. Do you mean digest 5' end, fill in, purify, then digest the other end purify and finally ligate? If so, I agree totally, that is the cleanest way to do the job.
Swanny: I understood the same as you. Isn't it a great idea? It's the first time I get a great real solution here in bioforum.