I can not get colonies at all time. So I run my ligation product. Please help me to fix my problem. COLONY come on!
Also, I have some question.
1. I design my primer with two REs site, and I design extra 3 bases at the end. I am not sure the 3 bases were enough to Xho1 and EcoR1. NEB website shows 50% efficiency with three bases.
2. Because the two REs was so close in my vector, i.e., Xho1 (978), EcoR1 (931). So I just wonder if I need to do separately digestion step to prevent from the two competitive REs reactions. And I have done that, the attachment showed the result.
3. I also do one step, digest the pcr product with one enzyme, then put the ligase, if both enzyme and ligase were worked, pcr bands should be doubled. According to the gel, I found my PCR product with EcoR1 digestion was slowed migration then pcr product with Xho1. I think the band should locate in the same position. Do I need change the new REs?
4. Do I need stop now? Redo it? Or I can do next transformation?
Ps: Gel result has been attached. Lane 2 and lane 3 were not so clear because I just use 20 ng vector for 20 uL ligation system, then I just load the 5 uL on the gel.