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

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

Post by Newbie » Wed May 31, 2006 3:13 pm

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!

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Post by oppox » Wed May 31, 2006 5:26 pm

The thing that manuals warn you about is not using too much insert if its chloroform extracted and then its the volume of PCR product that could mess it up. Ive seen manuals suggest like a molar ratio of 10:1. But since your insert is so small the volume gets very small, im taking 1 microliter insert, but mine is around 750 Bp.

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Post by Newbie » Wed May 31, 2006 9:21 pm

So, using 1ul insert and vector has worked for you? Is it in a 20ul ligation reaction? Do you mind if I ask what concentration of vector and insert you start out with before and after purification?

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Post by oppox » Wed May 31, 2006 10:43 pm

Sure, yea I took 1 microliter of vector and primer, my primer is about 700 bp and the vector about 4000, the conc was somewere around 50 ng/microliter of each. This makes it roughly a molar ratio of 5:1 (in a 10 microliter ligation sample) and it worked. So try diluting your vector if u think the volume u take is too small. If u can, you can also try out with different molar ratios. But as I said be careful if u have a chloroform extracted insert, if u dont I cant come up with anything else that could mess up the ligation. Though you cant hold me to that :wink: . good luck

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Post by Newbie » Fri Jun 02, 2006 4:42 pm

Alright I will try it out...but if I dilute my vector I will need even less of the insert right which means less volume...but I'm trying to increase the volume of the insert while keeping the volume of the vector around 1ul...I guess I can always dilute my insert as well...we will see... :lol:

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Post by oppox » Sat Jun 03, 2006 12:47 am

oh yea well....I ment try dilute your insert...hmmm I must have been real tired when I wrote that reply hehe :roll:

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Post by tranjo » Sat Jun 03, 2006 3:21 pm

Try to do this empirically with a range of ratios. I think it's difficult with such a small insert to get good optimization.

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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Post by Newbie » Mon Jun 05, 2006 3:29 pm

O you are right! I didn't even about that yet...it would be hard to see but as of now I'm having enough trouble just getting some colonies...one step at a time I guess...

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Post by oppox » Wed Jun 07, 2006 7:31 am

Another thing, what cells are u transforming it in to?
Is the ligation mix allright?
What restriction enzymes do u use?
Do u know if the vector has been cut as it should be?

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Post by Newbie » Thu Jun 08, 2006 5:05 pm

I am transforming into DH5alpha competent cells, I think the ligation mix is alright...I'm trying it with fresh buffer and ligase in a 20ul ligation mix at 22 degrees for an hour now at various molar ratios. I re-made a fresh batch of insert and vector both around 30ng/ul. If I used 1ul of both insert and vector though I would have 70:1 molar ratio which seems a bit high. I'm also trying it using a bit more vector and insert, like 60ng vector and 8ng insert for a 10:1 ratio instead of say 30ng and 4 ng. The seems to be cut properly from the gel I ran comparing to uncut, I cut with XhoI and EcoRI. Although, I haven't checked if my inserts have been properly cut since the difference is only a few nucleotides between cut and uncut.

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