Transformation background problem - high background in negative control (Aug/24/2010 )

Hi everybody,
here is my problem
I tryed to ligate annealed oligos into a digested plasmid. Plasmid carrying a ampicillin resistance was single digested (BsiWI), CIP and ligated with my phosporilated annealed oligos, but I got hundreds of colonies in my negative control as well (ligation without insert). In order to tackle the problem I tried to overdigest (overnight) the plasmid, heat inactivate and gel purify. I transformed it into competent cell (16 ng) without even adding any ligase (just the linearized plasmid) and again hundreds of colonies. I checked for water contamination, and ampicillin plates, but the were both fine, I used two different strains of competent cell(DH5alpha, TOP10) and again same results. In the end I even tryed to repeat the all operation using this time a plasmid double digested with two different enzymes (EcoRI, AgeI), but again same results. I can guess that a small fraction of undigested plasmid could be always left (even if after a double digestion should be really minimal) but at the most I should get a couple of colonies not hundreds, or I can even say that bacterial cell could still religate the cut vector but the efficency should be very low not so high (and then even not compatible ends?). I wonder what am I missing here? any help appreciated.

Fabio

Do you purify your digested plasmid on gel?
Otherwise, you allways will have trace amounts of undigeted plasmid which is enough to override the transformation.

Fabio on Tue Aug 24 09:56:40 2010 said:

Since you get lot of colonies on negative, CIP did not work properly, did you try new CIP enzyme you can aslo try Tsap or SAP it works fine
try to solve the probem with negative control then go for the ligation

Do you gel visualise your digested vector? The first thing I would try is either testing the enzyme or buying a new vial.

Thanks all for the replys.
The enzyme is brand new and the digestion was done strictly according to manufacture recomandation.
The plasmid result totally cut on an agarose gel (comparing it with uncut DNA).
I am not sure is a CIP (or SAP I tried both)problem since I even tested DNA WITHOUT adding ligase, even double digested DNA! Now I wonder is possible that bacteria cell can religate linear plasmid?

Thank you again
Fabio

I have encountered this problem several times

What i would do cut the plasmid take an aliquot
and do the CIP or SAP treatment, heat inactivate the SAP or CIP(although CIP is not easy to heatinactivate SAP is)
You should do the pcr clean up or gel cleanup after the SAP
Then do transformation on both before and after CIP treatment
Compare the plates
If your unCIP treated has many colonies, your CIP treated plate should not have anything
Then you can be sure CIP is working well

I suspect the problem is not with CIP or SAP, but rather incomplete cutting of the plasmid with the restriction enzyme. Uncut plasmid transforms very efficiently. You could cut longer, or cut less DNA in the reaction, or cut a second time. I don't know how efficiently the weird enzyme you are using cuts.

Ok Ranvi I did your little experiment.
2 different plasmids digested in parallel (A and B ) with the same enzyme (BsiWI), then SAP.
TRansformation of equal amount (11ng) of DNA for unSAP and SAP DNAs, bear in mind NO LIGASE involved in this experiment.
REsults
A unSAP 46 colonies; A SAP 0 colony
B unSAP 96 colonies; B SAP 4 colonies

It' s evident that dephosporilation works(as phage434 spotted correctly). But I have a HUGE doubt....why I do have colonies in unSAP samples after all??
SAP can work only on already cut plasmid or maybe even on nicked, preventing it to be "transformed correctly". If I had uncut DNA after the restriction digest it should be transformed even after SAP treatment, and this does not happen (So even the restriction enzyme is working). Hence the colonies seen from unSAP samples can come only from already cut plasmid. So my big question for all of you guys is "can cut DNA be transformed?" "can bacteria ligate cut DNA?" "what will happen if I use double digest plasmid?"...."what am I missing? where my weird teory will fall apart?"

Thanks all

Yes, bacteria are very good at repairing pieces of DNA. If there is significant compatible overlap or homology between ends, then repair mechanisms can fix up linear dna and make transforms. Double cuts, leaving incompatible ends, solves many of these false positives.

I guess that even double digest DNA, if the two ends have some degree of compatibility, can still produce the same problem.
Thank you very much