Cloning problems i.e. Ligation - (Aug/31/2010 )

Hi,

I'm back again. I am still in cloning hell and maybe one of you will have some suggestions for me. I have my PCR generated insert in a TOPO vector. I want to ligate the insert into the pcDNA3.1 (+) vector. The insert is 3kb and the vector is 5.4kb. I cut about 5ug of vector and add in 2.5uL of my Acc651 and 2.5uL of Xhol and incubate these for approximately 2-3 hours. When I run a gel, it looks like my vector is cut because I also run single digests of the respective enzymes. I do not dephosphorylate the ends and have never needed to do this. I gel extract and purify both insert and vector. I then ligate using 1uL of T4 DNA ligase for 1 hour at room temperature. I do molar concentrations of my vector and insert i.e. I add about 50ng of vector and use a ratio of 3:1 of insert to vector and calculate the amt of insert to add based on the size of the insert and vector. However, I am getting almost the same # of colonies on my vector only and vector+insert plates. When I sequence the vector+insert colonies, I am seeing that there is no ligation, it is just vector. I am at a loss. Can anyone point out sthm I am doing wrong in the summary I have written up. Help is really needed.

How close together are the two cut sites of your vector? Perhaps after one enzyme cuts, the other enzyme is unable to cut because the cut site is too close to the end of the (now linear) sequence.

Ropa,

I would suggest you incubate your ligation mixture overnight at room temperature. I have always done that with T4 Dna ligase and usually, they have worked out fine.