Cloning after amplifying with Taq polymerase? - (Apr/09/2007 )
I want to clone a cDNA 1.4kb in a T/A clone. I could amplify that with Taq polymerase, but not with a proofreading system Expand High Fidelity PCR system of Roche. Both methids give T/A ends, but i thought to try the Roche system because it has the prooreading capability.
I am thinking to proceed to the cloning with the Taq amplified cDNA, which i purified out of an agarose gel.
Do you think the chances that i am having errors in my sequence are high?
Does it worth to spend more time trying to get my product by using The Roche system?
Anybody, who tried this system in the past can give me some tips on the PCR reaction? The AT that i used was 58oC (according to annealing temperature of my primers), will it make any difference if i decrease the AT?
Thanks in advance piki
You'll reduce the likelihood of cloning a fragement with mutations if you go with the high fidelity enzyme and probably save yourself time and headache later. But, if you have sequence to compare against and can sequence your cloned fragments, Taq's not that bad for a 1.4kb amplification. Taq can also be ideal for troublesome amplifications because it is more robust. However, reducing your AT too low will create mispriming and non-target fragment amplication.
Well, in my lab we came to the conclusion that, although a proof-reading polymerase is more expensive, that extra cost may be worth it. Using a proof-reading we usually just send 1/2 clones to sequencing, and with a non-proof-reading sometimes we needed 10 or more clones. It depends on how much you pay for the sequencing...
Some people use both polymerases in the pcr mix in order to have the proof-reading capacity and also a good amount of product. Never tried it myself though, I usually have a good amount with the proof-reading we use.
I wouldn't recommend using Taq for cloning. You'll have headache later. Better use Hi Fi Taq. If your Tm is 58C, you could try using a Tm at 55C. Do a touchdown from 60C or 65C if you're concerned about non-specific bands.
Use Taq for cloning if you are using TA cloning or UA cloning method. Or else you have to cleave out the overhang A with Klenow fragments or something like that.
Try to use those proofreading polymerase that doesnt give you overhang A.
Thanks a lot for all your comments and recommendations.
I found out that we have Vent prolymerase in the lab, which is type of high fidelity Taq.
About the last comment..... I thought that it would be more difficult to clone something (esp. when we talk about sequences) larger than 1kb that has blunt ends. I expect that TA cloning will help the sequence to get inserted into the clone.
What do you think about that?