Hi all,
I have a few questions about ligation
1)Could you explain to me why you need an excess of insert compared to the vector?What is the “chemical principle” of this reaction?
2)In my particular case, I have to ligate two inserts into the same vector but I don’t know how to choose the right ratio because the plasmid seems much less concentrated than the inserts. I attached a picture of the gel I had after restriction digestion and gel clean-up. The size of the vector is approximately 7 kb and the inserts are approximately 750-800 bps. The first two bands in the gel are the two inserts and the third one(the least bright)is the vector. One thing I did not understand is how to relate the length of DNA with the amount you have. The longer DNA you have the less amount you need to take?Is it right? So, if you can suggest the ratio to use according to the gel I have and explain to me your choice, it would be great because I am quite confused.
3)Also, how do you estimate the ng of plasmid you have based on the gel? For example in my case I know the ladder is not well separated but just to understand I make an example..if the plasmidic band was at the same level as the ladder band of 100 ng/band and I have loaded 5 ul , it would mean that I have a concentration of 20 ng/ul? What if the band is brighter or less bright?The ladder I used is that on the link.
http://www.bioline.com/sg/hyperladder-1kb.html
Thank you very much
Hello Silvia,
generally a molar ration of 3:1 (insert/vector) is considered a good starting point for setting up a ligation reaction. For doing the calculations you can use this site: http://www.promega.com/a/apps/biomath/?calc=ratio
Regards, Christian
in regards to ligation, always it is recommended to used more ratio of insert to that of vector thus reducing the chances of self ligation of vector (if u r using just one restriction enzyme). Usually 5:1 and 10:1 ratio works for most of the ligations. This also increases the probability of ligation of all desired fragments into the vector.
Use vector:Insert ratio 1:3/5/7/10 or keep 0.2 pmol end: 06 pmol end. keep in mind to use your vector at least 50-100 ng for good efficiency.
I am not sure if you have to do three pieces ligation into one recombinant molecule or two independent ligations. Typically, one vector plus one insert is straightforward strategy, while considering aspects of blunt ends, one vs two separate cohesive ends generated by restriction enzyme(s), and the dephosphorylation of the linearized vector to enhance recombinants. Molar ratios aspects are stated by others above.
For three way ligation, molar ratio of the two inserts should be equal, whereas preferably the dephosphorylated vector may remain at roughly 1/3rd amount. One may consider ligating the 2 inserts first, and then adding the vector to see if helps the situation. While working with bigger amounts, one may physically separate such ligated inserts and purify to set up second ligation with vector. This strategy may be useful when other options are not optimal, e.g performing blunt end ligations or one restriction enzyme linearized vectors.
Finally, one other important aspect is the ligation reactions themselves. Different ligation kits allow fast (5-15 minutes) at room temperature, and are popular choices. However, other factors including sizes of the inserts, ligation reaction volume, choice of the transformation host and their efficiency of transformation exist and stability issues may also arise and one has to troubleshoot the problems.
Generally the ration of vectors:inserts depends on the specific reagents you are using. Normally it varies from 1:2 to 1:3 in most cases.
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