Cloning problems i.e. Ligation
Posted 31 August 2010 - 01:57 PM
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.
Posted 31 August 2010 - 03:12 PM
Posted 31 August 2010 - 09:20 PM
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.
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