Well I usually use a calculator to help out
. Let me review a typical cloning reaction.
Suppose I want to clone a sequence of 1.5 kb into the TOPO vector (3.9 kb). I am using this for downstream applications, so I want to minimize any possible mutations by using a proof-reading polymerase (Phusion, Pfu, etc.).
So, I run PCR and I have a nice product, which I have purified (gel purification, etc.) and I measure its concentration. Let us suppose that my 1.5 kb purified PCR product has a concentration of 50 ng/uL.
Now, I need to add the 3'-adenine overhangs in order to perform TA cloning. To do this I use a standard Taq with 10 mM dATP (or 10 mM dNTP mix works fine) and 10X Taq Buffer.
I want to clone in my 1.5 kb product in a 1:1 molar ratio with the 3.9 kb TOPO vector. My TOPO vector is 10 ng/uL. So:
(10 ng TOPO) * (1.5 kb PCR product)
------------------------------------ = X ng PCR
(3.9 kb PCR product)
This means in my TOPO reaction, I will need to assemble:
10 ng TOPO + 3.8 ng PCR product.
So to simplify my life, I will plan my 3'-adenine Reaction to have a final concentration of 3.8 ng/uL. Thus my TOPO reaction will be:
3 uL H2O
1 uL Salt Solution (with TOPO kit)
1 uL 3.8 ng/uL Taq-treated PCR product
1 uL 10 ng/uL TOPO vector.
6 uL total.
If I want a 25 uL Taq reaction with a final concentration of 3.8 ng/uL, then I need to seed it with 95 ng of purified PCR product. Since my original PCR product concentration above was 50 ng/uL, I will use 1.9 uL:
9.6 uL H2O
12.5 uL 10X Taq Buffer
0.5 uL 10 mM dATP (or 10 mM dNTP mix)
1.9 uL of 50 ng/uL Purified PCR product
0.5 uL Taq
I mix this up, spin it down and incubate it at 72C for 10 minutes. Then I assemble my TOPO reaction as above, using 1 uL of Taq-treated PCR product.
The whole point is so that the molar ratio of TOPO vector to Taq-treated PCR product is 1:1. This produces the most efficient ligations and works for me every single time, with almost all white colonies after transformation.
If you do not need to use this for downstream cloning and you have been using Taq polymerase all along, then the addition of 3'Adenine is unnecessary.
Suppose then that I have 50 ng/uL of purified 1.5 kb Taq-amplified PCR product. Well, my optimal ratio is:
10 ng (1 uL) TOPO : 3.8 ng PCR product.
Now, I won't be able to pipet 0.076 uL, so I dilute my PCR product down to 2.5 ng/uL (20X dilution) in water, or Tris buffer or something. Then I can use ~1.5 uL of 20X diluted PCR product in my TOPO reaction:
2.5 uL H2O
1.0 uL Salt solution
1.5 uL 20X diluted purified Taq-amplified PCR product
1.0 uL 10 ng/uL TOPO vector
6 uL total (5 minutes incubation @RT).