Creating empty vector - (Jan/05/2009 )
Hi everybody,
Can anybody suggest me about creating an empty vector. I am using a TA-TOPO cloning vector. I have inserted and cloned a 2.2 kb pcr product without any problem. But I need to prepare an empty vector as transfection control. As I have mentioned this is a TA-TOPO cloning vector i.e. comes as linearalized with t overhangs and TOPO isomerase. Please help me to create an empty vector i.e. circularize the pplasmid.
Thanks in advance.
Ashraf
you could use klenow enzyme to create blunt ends, and then ligate.
try to just transform the linearized topo-ta plasmid into your bacteria.
In my experience, you can generate background colonies of topo-ta plasmids if you do not offer any pcr product to be cloned.
So, take the plasmid only - perform the same protocol and select.
The output won't be high probably, but you might well get some colonies. upon selection.
I don't how it works but I think in some rare cases the bacteria ligate the lineraized plasmid themselves.
Good luck!
In my experience, you can generate background colonies of topo-ta plasmids if you do not offer any pcr product to be cloned.
So, take the plasmid only - perform the same protocol and select.
The output won't be high probably, but you might well get some colonies. upon selection.
I don't how it works but I think in some rare cases the bacteria ligate the lineraized plasmid themselves.
Good luck!
I am kind of thinking that the TA vectors companies sell are not 100% efficiently made. So there are bound to be a lot of non-overhang molecules which will self ligate. Upon storage too, there will be many blunt molecules generated. Whatever the cause, you will get a lot of empty vector colonies!!
In my experience, you can generate background colonies of topo-ta plasmids if you do not offer any pcr product to be cloned.
So, take the plasmid only - perform the same protocol and select.
The output won't be high probably, but you might well get some colonies. upon selection.
I don't how it works but I think in some rare cases the bacteria ligate the lineraized plasmid themselves.
Good luck!
I am kind of thinking that the TA vectors companies sell are not 100% efficiently made. So there are bound to be a lot of non-overhang molecules which will self ligate. Upon storage too, there will be many blunt molecules generated. Whatever the cause, you will get a lot of empty vector colonies!!
Try a ligation anyway and transform into the bacteria, colonies will usually come up (in my experience)
In my experience, you can generate background colonies of topo-ta plasmids if you do not offer any pcr product to be cloned.
So, take the plasmid only - perform the same protocol and select.
The output won't be high probably, but you might well get some colonies. upon selection.
I don't how it works but I think in some rare cases the bacteria ligate the lineraized plasmid themselves.
Good luck!
I am kind of thinking that the TA vectors companies sell are not 100% efficiently made. So there are bound to be a lot of non-overhang molecules which will self ligate. Upon storage too, there will be many blunt molecules generated. Whatever the cause, you will get a lot of empty vector colonies!!
Try a ligation anyway and transform into the bacteria, colonies will usually come up (in my experience)
i have done transformation with the plasmid only, and now waiting for the colonies to form, finger crossed.
thanks.