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T- vector clone


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3 replies to this topic

#1 weixiang

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Posted 27 September 2004 - 05:29 AM

dear,


I have got a 9.6kb with takara LA taq,I'm wanting to clone this insert using takara pMD-18T easy vector cloning system. I've tried many times,but the result is very undesirable!i am vrey confused.Is that the T-vector have not clone long DNA product?or others?If my DNA product have complex construcure?I do't know.when t have try it,i 've gel extracted it and eluted in 50ul water,ligation was done at 16oC overnight.then tranformation with complete JM109.
Do you spend your time tell me? what limit long PCR product clone to T-VECTOR?
i show my best whises to you !thanks very much!

#2 weixiang

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Posted 04 October 2004 - 03:59 AM

I succesed makeing a TA clone with my 9600bp pcr product.
thanks for all!

#3 drjulianz

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Posted 08 October 2004 - 12:15 AM

first ,congratulations! i am interested in a ligation of such a long fragament to the vector. Did you find the reason why the insert can not be cloned in the vector previously? and what is the ratio of your insert to fragment?Thanks

Edited by drjulianz, 08 October 2004 - 12:16 AM.


#4 pwqin

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Posted 19 October 2004 - 12:01 AM

I have PCR a 4.5kb fragement using TAKARA EX Tag and also using PMD-18T vector to clone. I have gel extracted my fragment and add 5' phosphorous and extend using klenow fragment, then ligate at 16 degree overnight. Transformed DH5alpha many times, but the result is disappointed. can you tell your precious experience. Thanks in advance.




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