I tried CircLigase a long time ago but it did not work on my self-made test DNAs, and only circularized the positive control DNA provided by the manufacturer. My self-made DNAs were 5'-phosphorylated (and doped with 5'-radiolabeled DNA, so at least for that fraction I am 100% sure that there was a phosphate group). Also, T4 RNA Ligase was able to circularize some of my test DNAs. In the end I found another protocol for my specific problem, but I am still puzzled by this result, as CircLigase is used with success by a lot of people doing deep sequencing. So I was wondering if anyone of you has studied the influence of sequence itself and/or length on the circularization activity of this enzyme. Thanks!
Have you tried different Mn++ concentrations? This group did some optimization and found that different concentrations were better for different size DNAs.
Some other tinkering found nucleotide bias that promoted circularization
http://www.promega.com/~/media/files/resources/conference%20proceedings/ishi%2019/oral%20presentations/nunez.pdf
http://www.sciencedirect.com/science/article/pii/S1872497311000871#
Hi Marie-Luise Winz ,
It's a long time since you asked this question but may I ask how long were the fragments you were trying to circularize? Did you solve you problem?
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