## Vector is 80X larger than insert, what molar ratio to use?

**Moderators:** honeev, Leonid, amiradm, BioTeam

### Vector is 80X larger than insert, what molar ratio to use?

Hi everyone,

I am doing some shRNA cloning with an 8kb vector and 110bp insert and cannot for the life of me get any colonies. One problem that has been plaguing me is that after calculating the molar ratios I find that I need much less insert than vector, making the volumes a bit awkward. I think mathematically it is correct but practically it seems odd to me because we usually use more insert volume than vector. I calculate it like so for a 3:1 ratio of insert to vector:

(mass of vector/length of vector)/(mass of insert/length of insert) = 1/3

Rearranged and with the values it would be:

(50 ng vector X .11 kb insert) X (3/1) = 2ng of insert

8kb vector

So we would only need 2ng of insert per 50 ng of vector? That seems too little! That means I would only need like 0.15ul of insert for 1ul of vector (since my vector is 50ng/ul and my insert is around 15ng/ul) Do you see what I mean? It was suggested to me in this case to use a higher molar ratio. My post-poc even said to use as much insert as possible, like 7ul insert and 1ul vector in a 10ul ligation reaction...that is way too high isn't it??? That would make the molar ratio about 150:1! Is that really efficient to use more insert? But then how would I deal with the volume issue? Or do I just ignore the fact that the volumes seem strange and use a very small amount of insert? Please advise!

I am doing some shRNA cloning with an 8kb vector and 110bp insert and cannot for the life of me get any colonies. One problem that has been plaguing me is that after calculating the molar ratios I find that I need much less insert than vector, making the volumes a bit awkward. I think mathematically it is correct but practically it seems odd to me because we usually use more insert volume than vector. I calculate it like so for a 3:1 ratio of insert to vector:

(mass of vector/length of vector)/(mass of insert/length of insert) = 1/3

Rearranged and with the values it would be:

(50 ng vector X .11 kb insert) X (3/1) = 2ng of insert

8kb vector

So we would only need 2ng of insert per 50 ng of vector? That seems too little! That means I would only need like 0.15ul of insert for 1ul of vector (since my vector is 50ng/ul and my insert is around 15ng/ul) Do you see what I mean? It was suggested to me in this case to use a higher molar ratio. My post-poc even said to use as much insert as possible, like 7ul insert and 1ul vector in a 10ul ligation reaction...that is way too high isn't it??? That would make the molar ratio about 150:1! Is that really efficient to use more insert? But then how would I deal with the volume issue? Or do I just ignore the fact that the volumes seem strange and use a very small amount of insert? Please advise!

Also, hope you have a good way to screen it cause if you run a gel of digested mini-prep samples, that insert will contribute very little, and thus be very hard to see on a gel!

Best, J

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