insert to vector ratio - (Jan/31/2007 )

Hi everyone,

I am new to this discussion. I am having problems with my ligation, i have an insert which has overhangs for Nco1 and Bamh1 and i trying to ligate this into my vector which has the same recognition sites, i would like to know if i am correct in my calculation to do a 3:1 insert to vector molar ratio.

insert size-74bp

vectore -3000bp

mass of vector= 500ng so how many microlitres of vector and insert do i need to do the ligation.

kindly help me with this


Hash

I would use 20ng of vector and 1.5ng of insert for 1:3 ratio.

Normally a 5:1 ratio of insert:vector works best for 75 - 100 bp inserts and 3000 bp vectors. While a 10:1 is still safe, going above the 10:1 ratio will generate a certain number of double-insert colonies.
If the restriction sites you are using in the vector are both in the multiple cloning site, then you can shortcut the purification of the vector afterwards by using a 30K MWCO cartridge. Most of the small fragment will pass through the membrane and the linear vector will remain in the membrane cartridge. This is considerably faster than gel-purification, and works surprisingly well.
Of course, I always recommend that you simultaneously dephosphorylate your vector during the restriction cuts with SAP.

A caveat about cutting the insert. 1 ug of a 74 bp fragment contains 1.25 e 13 molecules. That means you have 1.25 e 13 restriction sites as well. 1 ug of 3000 bp vector only contains 3 e 11 molecules. You have almost 42x as many restriction sites in the insert reaction as you do in the vector reaction if you cut 1 ug of each. Keep this in mind when you set up the digests. If you cut the same number of molecules of both insert and vector, then when gel analysis tells you that the vector is cut to completion, you can normally assume that the insert is also cut to completion.

give a try with 1:10 ratio