Timeline for Can someone link me to resources on the efficiency of sticky end ligation?
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| Mar 17, 2015 at 20:38 | comment | added | mdperry | The enzymatic reaction requires adenosine triphosphate as a co-factor, and Mg++ (as well as a dilute buffer). The substrate is double-stranded fragments of DNA possessing 5' phosphates. Blunt ended DNA fragments can be ligated to each other, and this reaction is enhanced by adding polyethylene glycol. However DNA with complementary overhangs are ligated much more efficiently. The reaction is at least trimolecular (2 DNA ends + 1 protein) so the reaction volume affects the concentration and therefore the rate. So how long do you want to let the reaction proceed? | |
| Mar 17, 2015 at 20:23 | comment | added | The Obscure Question | I guess I want to know what the maximum possible efficiency of religation is. | |
| Mar 16, 2015 at 23:31 | comment | added | Katz | He is poposing to digest with restriction enzime this gene and check after ligation the % of blue/white colonies (of course after restriction and ligation E.col are transformed).If all ligation process happened without introducing any indel, the gene should be functional and the colonies blue, and the % of blue colonies serves as a readout of efficiency | |
| Mar 16, 2015 at 23:30 | comment | added | Katz | Altought it it is could be more digested, I think that the response is not unanderstandable: it refers to the prototypical "blue-white assay" using the LacZ gene, which encodes beta galactosidase and this digests X-gal making bacterial colonies blue.(the word "cfu" stands for colony forming unit) | |
| Mar 16, 2015 at 23:20 | comment | added | canadianer | Yes, this is entirely understandable. | |
| Mar 16, 2015 at 22:56 | comment | added | AliceD♦ | Welcome to Bio.SE and thanks for sharing your answer. Please avoid using acronyms and unintelligible jargon. I have a biochemical background (ages ago since last using it admittedly) and frankly I don't understand this answer. | |
| Mar 16, 2015 at 22:26 | review | First posts | |||
| Mar 16, 2015 at 22:57 | |||||
| Mar 16, 2015 at 22:23 | history | answered | mdperry | CC BY-SA 3.0 |