Self-circularization problem with pUC19 - (May/08/2007 )
Hi all, i have this problem of self circularization of linear pUC19 digested with BamHI.
The concentration of linear pUC19 according to nandrop is 1532.76ng/ul
From this concentration i took 10ul. I add : 5ul 10x T4 Ligase Buffer Fermentas
34ul DNAse free water
1ul T4 Ligase Fermentas (5u/ul)
I incubate this at room temperature for 2hrs.
Before I run it on an agarose gel, the concentration according to nanodrop is around 890ng/ul.
However, when i run it on a 1% agarose gel, I couldnt see any band at all! Does anyone have any idea whats happening to my pUC19?
To visualize the DNA following ligation, the ligase must be heat killed. Otherwise, it sticks firmly to the DNA, shifting the bands. Do not use quick ligase, and heat kill your ligation reaction for 20 minutes at 80C, then run it on a gel. Your ligation reaction is being run at very high concentrations. This favors concatamers rather than recircularization. If your goal is to recircularize, then you should be doing these reactions at more like 10 ng/ul or less. Depending on your cohesive end GC composition, you will also likely have more success by incubating the ligation at 16C for an hour. You should also wonder where the extra "DNA" comes from! 10 ul*1532 ng/ul is not the same as 50 ul*890 ng/ul. Think protein.
If both ends have sticky end, dont really have to use ligase right? I mean... I do understand it will enhance the efficiency of ligation.
Wow.. you have a very high yield of your plasmid. If you use 10 ul meaning, you have around 150 mg of plasmid? I agree with phage434, way too much.
Another possibility maybe it is your electrophoresis problem. Just taking a guess.
what about using phosphatase
Even if the ligase shifted the band, i should be able to see some band in the gel. But i see nothing, i suspect my plasmid is actually contaminated. The A260/280 ratio is around 2.06. Probably due to RNA? I think the concentration is less than it actually. But ill try heating the ligation reaction after the incubation.
Run a gel without doing a ligation. This will tell you roughly how much DNA you have and how long it is. This is an excellent idea whenever you have ligation problems, to double check that the input DNA is there, and of the right length.
how u understand that you are facing self circularization problem???
DNA conc is really high as others said.
you must do CIAP treatment before ligation to prevent self circularization