serious problem in cloning
Posted 21 March 2010 - 03:57 PM
I check the restriction enzyme, purification kit , ligation kit and transformation , every steps. try to figure out whether there is some contamination. but still have not solve the problem.
1. I change new restriction enzyme.
2.all of my lab-mates use the same purification kit. it should be fine
3. I change new ligation kit.
4. I did negative control for the transformation. (just vector , no pcr product). The plate is clean, no colonies there.
5. the plate should be fine, as others also use my plate with amp.
Now , I have no idea what's wrong .
Is there anyone can give me some suggestion?
Posted 23 March 2010 - 08:39 AM
2. The insert might be incompatible with the cells - for example too CG rich - if so try another competent cells suited for this.
3. There can be non-optimal vector to insert ratio in your ligation reaction. Try 1:1 as well as 1:3 or so.
Posted 23 March 2010 - 04:46 PM
1. Treat some DNA ladder with T4 ligase for ~20 minutes at RT and run on a gel. The ladder should be shifted to larger sizes. If it isn't your ligase is the problem.
2. Similarly, treat your digested PCR product with ligase for ~20 minutes, and run on a gel. You should get a ladder, or at least, your sample should be more than 3x the PCR product size. If you only get dimer-sized product, one of the enzymes is notworking. My bet is that it's the SalI, because the NEB website says that SalI and PCR don't work well together (no explanation of why). You can, of course, do the same thing with your digested vector.