I am interested to know whether a 1:3 molar ratio is ok with a 5.4 kb vector [5.4 kb after double digestion,(actual size of vector is 7.2 kb) ] and 600 bp insert DNA ?
Is there any relation for molar ratio, with respect to size of Insert and vector DNA?
How to choose appropriate ratio on the basis of size of Insert and Vector DNA?
I appreciate that...great...!! The concept is to optimize the meeting of vector DNA ends with insert DNA ends during ligation reaction. Usually the vector will be larger than the insert. So when you take 1:3 ratio of vector:insert in terms of nanograms, then the vector number will be far, far less than aimed. so that based on size of vector and insert, the molar ends are calculated in the calculator. Based on the concentration of your DNA preparation + size, the calculator gives you how much vector and how much insert you should take for a 1:3 ligation reaction. Mostly this stands good for cloning in vectors that were digested using single restriction enzymes to reduce self-ligation of the vector. The insert ratio can be even higher to reduce self-ligation of the vector. When it is directional two RE digested cloning, only the molar ends matters and 1:3 ratio is sufficient. Once you know the basis, you may use the calculator..!!
Use this...Simple..!!
http://www.insilico.uni-duesseldorf.de/Lig_Input.html
Thank you jinesh. I do not want to use calculator , I am interested in concept behind it , if possible could you please share it
I appreciate that...great...!! The concept is to optimize the meeting of vector DNA ends with insert DNA ends during ligation reaction. Usually the vector will be larger than the insert. So when you take 1:3 ratio of vector:insert in terms of nanograms, then the vector number will be far, far less than aimed. so that based on size of vector and insert, the molar ends are calculated in the calculator. Based on the concentration of your DNA preparation + size, the calculator gives you how much vector and how much insert you should take for a 1:3 ligation reaction. Mostly this stands good for cloning in vectors that were digested using single restriction enzymes to reduce self-ligation of the vector. The insert ratio can be even higher to reduce self-ligation of the vector. When it is directional two RE digested cloning, only the molar ends matters and 1:3 ratio is sufficient. Once you know the basis, you may use the calculator..!!
Hi, you can find some tips and advice for your question https://www.addgene.org/plasmid-protocols/dna-ligation/, http://bitesizebio.com/10203/ligation-optimization/ or https://ita.promega.com/resources/product-guides-and-selectors/subcloning-notebook/, where you can find the classical subclpning guide that it is very interesting.
In general the advice it is to try different ratio because it not exist a sort of formula to decided what is the best ratio regarding the lenght of insert and plasmid, but it is better to try different ratio. In your case, with a small insert, in my opinion 1:3 it is a good choice to begin with...
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Methods
- Cloning