Ligation after SalI & XhoI Digestion - (Mar/09/2009 )
Hi, I have a problem with this ligation:
pGL3-E1B digested with SalI, XhoI or both to remove the luciferase secuence, then insert a new secuence with cherry color, unfortunatelly, after the ligation with T4 ligase, E. coli or LONG ligases, the transformation of Top-10, Supercompetent or Able Cells is not working, even that my control (pUC19) are growthing, I'm thinking that probably my DNA it could be degradated after the ligation reaction, what do you think??? I must to say that, even I cut with single enzimes, I'm not able to observe religation (all the reactives are news) of the pGL3-E1B digested or the reinsertion of the luciferase in the Vector...
Sorry for my English, I'm trying to learn this new language...
It is unclear how you are doing your experiemnt. Do you gell purify your DNA after digestion? If not, you should.
The most important thing is to do proper controls!
During a ligation a negative control should be done. Cut you plasmid
and transfect this into your cells WITHOUT religating. This will tell you if your digest worked. It's called a no ligation control. If you get colonies after this, it means your vector isn't completely digested.
Your DNA is not degrading after transfection.
SAL1 is notoriously hard to achieve complete digestion with. You might want to try a different appraoch.
Also, are your control transfections with puc19 NOT growing, or are they growing?
You should work out conditions to get puc19 to work first. Chilling on ice for 30 minutes, followed by 60-90 seconds heatshock at 42 degress, add media, grow 1 hour then plate. This will work for every plasmid. If it doesn't then one of your components (media?) is off.
OK, how did you get your cherry insert? SalI has troubles with PCR products, also it's inhibited by nucleotides, and pH outside of 7-8. Also, supercoiled DNA needs 10x as much enzyme as linear DNA to cut well, so are you sure you have double-cut the vector? Can you give us a photo of the uncut, single-cut and double-cut vector?
Test your insert digestion and your ligase by a quick ligation. Take some insert (after you have killed the restriction enzyme, of course) and try to ligate it to itself for ~20 minutes at RT, then run a gel. You should have bands of monomer, dimer, trimer etc. Do the same reaction with some DNA ladder; the ladder should shift upwards in the gel. Again, a photo of your gel will help us.
If the DNA ladder shifts, but your insert only goes to the dimer, the ligase enzyme is OK, but one of your restriction enzymes is not working (my bet is on the SalI!). Try putting the PCR product through a cleanup step then re-digest.
Ur choice of enzyme combination is all the problem.....SalI and XhoI produces compatible ends......it is notoriously difficult to work with these combination. Using SalI and XhoI seperately or along with other enzyme never gives any problem. So use different enzyme combo, for eg: XhoI + something else!! or SalI + something else!! if situation allows.
Another important fact is once ends produced by SalI and XhoI ligates among them..it cannot be cleaved by either of the enzyme later.
I think ur vector backbone is religating/re-circularizing after cutting with SalI and XhoI...and the same wth ur insert.
Hope this helps.....if search the forum under cloning u will see user called clonetech (which was me) posted the same problem last year. Later I figured it out the hard way.
Hope this helps.