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cloning vectors - limited insert size (May/26/2006 )

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hi everybody, smile.gif

while i'm reading about cloning vectors,
this Q. comes to my mind:

why cloning vectors have limited size, i mean why these vectors accept DNA inserts only to a limited size?
plasmids foe example : why they can't accept more bp?

and how to know how many bp can a vector accept?! ph34r.gif


you can clone longer DNA within your vector but if you do so your transformation efficiency become less than you could get what you want.

when you buy a vector in company catalogue written for example: This vector can accept DNA inserts of up to 7.6 kb.



this is a question of basic microbiology. you must understand that all bacteria cannot be transformed in nature. some can be some cannot, depending upon their competency. even those that can be transformed, will accept external dna of a specified size, and this varies from vector to vector. e. coli is a bacteria that is not transformed in nature, and is hence not naturally competent, which is why we have to go through the process of making it competent. most cloning vectors are plasmids, and bacteria will accept plasmids of upto about 15 kb (vector+insert), with exceptions like the ti plasmid that naturally is 200 kb long but those too are specific forspecific bacteria like agrobacterium. for longer inserts in e. coli, uptoabout 25 kb, there are other vectors like phage lambda dna, since there the dna is entering the bacteria through a phage. still larger inserts can be put in through cosmids which are combinations of the bacterial plasmid and phage dna cos sites (cos sites responsible for dna packaging in phage head), thus cosmids with very large inserts, upto 45 kb, fool the phage and the rec dna gets packaged, only to be injected later into a bacterium.

hope this makes clear why vectors are size-limiting.



thanx akhshik, viv

you mean that insert size is crucial for packaging and hos cells...

but if i go crazy and clone a dna insrt larger than what the vector can accept, what will happen?


hey strawberry

go ahead, get crazy, do what you want, and let me know what happened.......


viv wink.gif


The limitation is not in how much DNA the vector can accept, but in the size that can be transformed into the cell.

There are, for example, several examples of megaplasmids known (for example, the 1,354,226-bp Sinorhizobium meliloti pSymA megaplasmid, and similar huge plasmids found in Agrobacterium tumefaciens, Brucella melitensis, and (perhaps) Vibrio cholera), but you'd never be able to transform them into E. coli...


QUOTE (strawberry @ May 27 2006, 05:01 AM)
but if i go crazy and clone a dna insrt larger than what the vector can accept, what will happen?

nothing special, maybe just by chance {very low} you could get the right transformant, one of my transformants once gots half of the gene from Right border of T DNA!!! may be you could get another strange things.

have fun.


so, the key here is the host in which we are going to transform our vector, right!


QUOTE (strawberry @ May 28 2006, 12:43 PM)
so, the key here is the host in which we are going to transform our vector, right!

hey, I use to work with adenoviral vectors, and we cloned the the complete adenoviral genome (50-60kb) into a "commun" low copy number plasmid like-pBR without any problem. We used E. coli to transform that plasmid. it's recommendable to use XL-gold or XL-blue E. coli strains, but I used Top10 too and it worked! (it's not recommendable to use this last atrain because the increase of mutations in your sequence or something like that).


the general event is a recombination inside the insert, giving you sthg strange. Ex: trying to clone a 7kb fragment, i obtain everytime sthg between 2550 and 2600... (over 50clones analyzed... blink.gif) but why? i don't know why !


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