cloning excised product in pGBKT7 doesn't work! - (Dec/17/2006 )
Since 2 months I am trying to clone a 1,4kb fragment, excised from pCR4 with SalI, in pGBKT7, opened with SalI and SAPed. Sounds easy as cake, but I get no insert carrying colonies out of my transformations. I am so desperate I started with colony lifts. To date I screened ~10000 colonies. I checked all my enzymes (they work great), I use commercially competent cells with 10^9/myg. The two pieces look good on a gel. i tried purifying/ not purifying each of the fragments, SAP, not SAP and still I get no positives out of it
please help!!!! I know this vector is a bitch, but it HAS to work.
thanx for every reply
I believe that SalI is a bad enzyme. It has a bad reputation in my lab. Any vector or insert from a vector cut by SalI will fail to ligate.
My suggestion would be to use some other enzyme.
hi there. thanx for reply.
but the thing is: i cloned a lot of stuff with salI and it always worked fine for me. i don't have anything against salI and never experienced that it produces no ligatable ends...
well, if you believe it is not SalI, then there are many other factors.
have you looked at the usual suspects?
- bad restriction enzyme.
- bad T4 ligase, ligase buffer
- too much UV
- over dephos
- wrong vector/insert
- lost DNA prior to transformation.
- nasty detergent leaking from the digestion buffer, killing cells (although, if you are seeing lots of colonies, this many not be a problem)
-PEG from quick ligase formulations must be removed, as PEG reduces ligation efficiency.
You could try different ligation conditions. 16 celsius overnight. 4 Celsius overnight. Could use quick ligase and ligate for several hours (2-3hr)
If you need to do colony hydridisation, something is wrong with the cloning.
Are are any of your other plasmids experiencing problems.. and should this be your sole plasmid, are any of your other labmates experiencing cloning problems. A quick way to find out if there is a problem with the lab common reagents.
If you are sure that all your reagents are working, welll I would say change the cloning strategy. Ligation as you know is a bit of a black art, somethings just don't work so one must change.