Dear all,
I'm in a problem with my insert and vector ligation. I'm trying to clone a 190 bp insert into a 2.9 kb digested MCS2 vector. I made restriction enzyme digestion and dephosphorylation at the same time. For this purpose, I used 2 ug MCS2 vector with two enzymes: 10 unit MluI, 10 unit NotI and for dephosphorylation I used 1 unit SAP at 37 °C 30 min for incubation and 70°C 20 min for deactivation. After gel prufication I had 11ng lineer DNA sample at the 1.7 260/280 and 1.4 260/230 OD ratios. I run my vector and annealed oligos in a gel and they looks like ok (Fig1).
Then I ligate them under these conditions,
4 ul 50 ng vector
1 ul insert (in different vector/insert ratios 1:1, 1:3, 1:5, 1:10 and 1:50)
5 ul 2X NEB T7 ligase buffer
1 ul NEB T7 ligase, incubate them 20min in room temp. (Fig 2)
But after exonuclease V treatment of ligation product, I lost all of them.(Fig 3)
How can I overcome this situation?
Any comment or suggestions will be really appreciated. Thank you all.
Why the Exonuclease V incubation after the ligation? Will you be ligating additional inserts into the vector? Is is possible that your DNA is not 'lost', but the concentration is too low to visualize on your gel?
Thanks for your all comments.
I use exo V for degrading non-ligated product. Thus, I don't go any further experiments if I couldn't see any ligated product in my gel. By the way, I solved my problem with increasing ligation time to 1 hour and excluding the dephosphorylation.
Thank you all.
1. Yes, you don't need to use 'Dephosphorylation' if you use two restriction enzymes for digestion. Dephosphorylation is used to prevent 'self-ligation' when one restriction enzyme is used.
2. It is interesting to know the use of exo V for degrading non-ligated product after ligation. But, even without using exo V, we can still see two large bands (one ligated supercoil, and one is the leftover backbone if not completely used) + insert, right?
3. By the way, does your insert (190-bp) has phosphate group?
Dear Yuan-Yeu Yau,
Sorry for long silence. The three bands is our inserted plasmid formation in this picture. And yes, we can still see the plasmid bands in gel without using exo V. By the way, our oligos phosphorylated with PNK.
Interestingly, our two different cohesive end,with GGCC (notI) and CGCG (mluI), ligated without the instert addition. Thus, I changed mluI with BamHI.
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