Cloning a large PCR product - trouble cloning large DNA (Apr/26/2005 )

I am trying to clone a PCR product amplified with elongase and is bigger than 12 kb.
My efforts to clone the same in pGEM3Z (with blue-white selection) have failed and also the TA-cloning kit did not result any positive clonings.

however, I do know that cloning per se is working as my control fragments of up to 5 kb are successfully cloned in TA cloning system and also about 3 kb fragments clones in pGEM3Z.

I would be thrilled to know if someone knows a magic vector/kit that can enable me to clone large DNA fragments and analyze the same in Escherichia coli and hopefully either have suicide vector system or some phenotypic selection to make it easier to analyze clones. If there is no possibility for direct selection either by survival of recombinant or phenotypic I would give a try to them as well as I am desperate to get this PCR product cloned for sequencing reasons, I would hate to sequence the whole PCR product from amplified DNA.

Aru

hi
do you mean by TA kit the kit that contains the topoisomerase? cause in my lab we use the ta cloning kit too but it does not contain the topoisomerase...

For cloning big fragments, i've heared long time ago about cosmids... but i don't know if it's a good sugestion as i know very few things about them...

Fred

Yes, I meant TA cloning kit. It did fail indeed for each repeat. I have however been told by tech. support that the quantity of the DNA (pcr product) must not be too high, gel purified and very fresh and that ligation reaction must continue for hours instead just 5 minutes as instructed by kit. This is like some sort of hidden charge by seller. I am going to give a try but I need to repeat PCR and am failing to amplify with good quantity.
Thanks for your query and suggestion.
Aru

Hi,

for cloning of large genomic DNA-fragments I would use the lambda red/recET-
system. A late review about cloning techniques is the following link:

http://www.genome.org/cgi/content/full/14/10b/2020



A system I have used in the past to modify large plasmids (> 45 kb) was the
following:

Datsenko, K.A. and Wanner, B.L. 2000. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc. Natl. Acad. Sci. 97: 6640-6645.


But I just generated knock outs, but with a few modification it should be able
to do also the cloning job, and: it's (almost) free for academia.



It is the same principle as the recET-system which was also used for subcloning
large genomic DNA-fragments. Have a look at this article:

Copeland NG, Jenkins NA, Court DL.
Recombineering: a powerful new tool for mouse functional genomics.
Nat Rev Genet. 2001 Oct;2(10):769-79.



Here's a site for a kit:

http://www.genebridges.com/web/technology/redet.htm



Bye,
Hennry