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[manueliux_06]

Hi, my english is not so good, I hope can explain correctly.

I'm trying to subclone expression cassette (3392 bp) of pHANNIBAL to pCAMBIA. I make digestions to pHANNIBAL with Not I to liberate the insert and pCAMBIA with EcoR I to to linearize the vector   approximately 3 micrograms of DNA. After the digested mixture is added  Klenow for fill-in reaction according indications of invitrogen which found that terminate fill-in reaction by phenol extraction.

  
And here is the problem because after the phenol extraction I quantified in NanoDrop and only get 1-2 ng /ul. This amount is not sufficient to continue the dephosphorylation with CIAP. Previously I cloned with cohesive ends and added CIAP to the digested mixture for dephosphorylate, then I run agarose gel and I purified bands with kit of General Electric. Finally I obtained (10-15 ng / ul) After I make ligation reaction and  the cloning fragment is done, but now,  I'm stuck with using Klenow because after phenol extraction I have little DNA.