T- vector clone - (Sep/27/2004 )
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!
I succesed makeing a TA clone with my 9600bp pcr product.
thanks for all!
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
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.