I hope someone can help me out! I normally have no trouble with cloning but for some reason my latest efforts just ain't working :/
I need to replace the promoter of one vector with another. RE sites to use are very few so this is what I have been trying to do:
1. Remove existing promoter with Xbal and NotI (get expected sizes so all good here).
2. Remove new promoter with StuI and MluI (again, expected sizes).
3. Gel purify bands.
4. Blunt ends with Mung Bean Nuclease and purify.
5. CIP-treat vector and purify.
6. T4 Kinase treat insert.
7. Ligate 16degC ON.
I have also tried a few variations of not CIP-treating/T4 Kinase treating but absolutely nothing has worked :/
Any advice would be greatly appreciated!