I really would like to know if sticky end ligation could potentially be performed with very high efficiency, and which factors influence that. However, I can't find any papers on the subject, even though I'm sure they exist. I was hoping someone would know of some literature.
$\begingroup$
$\endgroup$
2
asked Mar 16, 2015 at 14:50
-
2$\begingroup$ What exactly do you want to know? $\endgroup$– WYSIWYGCommented Mar 16, 2015 at 16:19
-
$\begingroup$ I want to know if there is literature that shows that sticky end ligations occurs without indels and that sticky end ligation can be done with around 80%+ efficiency. $\endgroup$– The Obscure QuestionCommented Mar 16, 2015 at 19:36
Add a comment
|
2 Answers
$\begingroup$
$\endgroup$
The best resource for troubleshooting ligations I found (and use frequently) is this NEB page. That is assuming you've already referred to the instructions provided with the enzyme you're using. In my case it's T4 Ligase, again from NEB. It's helpful to check the FAQs and the references listed on that page. Also, you can make use of their Molar Calculator tool on Ligation setting (see more from first link).
Good luck with high efficiency!
$\begingroup$
$\endgroup$
6
To measure the frequency of indels at the ligation site you can use a vector with a unique restriction site in the lacZ gene. With a colorimetric assay you can count the number of white cfu. Perfect ligation in-frame yields blue cfu
-
$\begingroup$ 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. $\endgroup$– AliceD ♦Commented Mar 16, 2015 at 22:56
-
1$\begingroup$ Yes, this is entirely understandable. $\endgroup$ Commented Mar 16, 2015 at 23:20
-
1$\begingroup$ 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) $\endgroup$– KatzCommented Mar 16, 2015 at 23:30
-
1$\begingroup$ 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 $\endgroup$– KatzCommented Mar 16, 2015 at 23:31
-
$\begingroup$ I guess I want to know what the maximum possible efficiency of religation is. $\endgroup$ Commented Mar 17, 2015 at 20:23