5 replies to this topic

[hobopottamus]

I am currently attempting my first 3-way ligation with 3 fragments of different sizes, 10.7kb insert, 1.8kb insert and a 5kb vector. I am having trouble determining how to calculate the optimum molar ratio to be used. I don't even know where to begin does anyone know of any articles or formulas that can be used to determine this?

thanks -phil

[phage434]

1:1:1
It has to be, since your ligase doesn't know or care what is in the different DNA fragments.

[Purzel]

phage434, on 14 February 2011 - 12:32 PM, said:

1:1:1
It has to be, since your ligase doesn't know or care what is in the different DNA fragments.

One quick question. Let´s assume the third piece is a relatively short oligonucleotide (linker). Would you chose the same ratio?
Thx

[perneseblue]

Yes I would still keep the 1:1:1 ratio.

But generally I would insert that oligo using PCR amplification of the adjacent DNA fragment. You can use 100bp oligos as primer..  only the 3' end of the oligo actually anneals to the template. This allows you to add 80bp via the 5' end of the oligo.

[Qundo12]

That's pretty much easier than I though before and I succeeded with the molar ratio of 1:1:1 recently, the efficiency is 8/10 clones. So, to make it simple to you, if you use 50ng vector, you should use 107ng of the first insert and 18ng of the second insert, if you want to change the amount of vector, just change the others by the same proportion. Good luck!

[hobopottamus]

Thanks everyone! I will let you know how it turns out I'll be starting this week