Hi All,
I meet a ligation problem. I digest my vector with EcoR1/EcoR5, and insert with Mfe1/EcoR5, after ligation and heatshock, there is no colony grow. I run a gel with my ligation products, it shows my insert didn't link with my vector. Although EcoR1 and Mfe1 was different enzyme, but they leave a same sticky ends, so I think they should ligation each other well, but result didn't. I have no idea which part goes wrong. Can T4 ligase ligation differernt enzyme cut with same sticky ends? if T4 can, I may have digest problem.
If sticky ends are compatible they will be ligated by T4 no matter which restriction enzyme you use to create the ends. Troubleshoot the general aspects of ligation, such as make sure that the enzymes are still working, DNA has good quality and quantity, competent cells are not dead, antibiotics are correct etc.
1. It is not surprising that you didn't see ligated product when you ran the ligation mixture on an agarose gel. This is because not more than 0.1% molecules used the reaction mixture get ligated.
2. No colonies, which means double digestion of plasmid worked (otherwise you would get colonies carrying plasmid without insert due to self ligation).
3. What is the source of your insert. If the insert is PCR product, then you may not know whether it is fully digested by the two restriction enzymes or not. If the digestion is partial, then it will effect the ligation.
However, if you released the insert from a plasmid by restriction digestion, then it may not be the reason for not getting colonies.
4. Ligation of sticky ends (produced by EcoRI/MfeI) is more efficient than blunt ends ligation. So you may need to use conditions (ligation temp, PEG etc) that are optimal for blunt end ligation to be successful in getting colonies.
I agree with the other responders. This should work. Indeed, even if your insert is not going in, you should still get some colonies from religation of the vector with the excised EcoRI/RV fragment. The fact that you get no colonies at all indicates that either your ligation has failed or that your transformation isn't working.
I would suggest the doing the following: Cut the vector with EcoRI and RV alone and check that you get religation. Do remember to include the cut but unreligated vector as a control and to standardise the amount of vector DNA.
Also, check the symmetry of the cut sites. There are two different possible placings giving a "3' overhang" and a "5' overhang" respectively. The sequence of the overhang may be the same, but the ends are not compatible.
On the other hand, if the supplier says that MfeI is compatible with EcoRI, you can be fairly sure that it is.
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