serious problem in cloning - (Mar/21/2010 )

My cloned my pcr product into pFaster vect, using the restriction sites of NotI and SalI. it works well. But recently , i always failed to get the positive clones. After ligation and transformation, I got some colonies on the Amp plate. But all of them are not what I want to get. They are also not the vector self-ligation. I really have no idea what are they.

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?

1. Are your competent cells OK? Do the posivive transformation control if you are unsure - transform some circular (supercoiled) plasmid.
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.

2 things to check.
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.

Good luck.