PCR optimization - (Aug/10/2005 )
Phage434 there are many reasons to optimise a PCR reaction that is working
1. To reduce the cycle number to enable more reactions to be performed.
2. To reduce the amount of expensive reagents used.
3. To improve yield if you need to make a lot of a particular product
4. To reduce non-specific bands and primer dimers.
5. To improve the robustness of the reaction so that is will work reliably on a wide variety of DNA samples.
6. To improve the reliability of the reaction so that others have a chance of repeating your work.
7. Self pride. You are a pretty poor scientists if you can't be bothered working out the best condition for your experiments
I am sure there are other reasons, but this is enough for now
DNA sequencing software
Foolish me -- I didn't realize my PCRs were art objects. I thought they were tools. I think your points are very valid if you are doing a many similar PCR reactions. I hardly ever do that -- every one is new. When this is true, there is strong motivation to make the first one work, not to spend a day or two optimizing the reaction. After a few rounds of making primers work following days of effort, I now take the attitude that a primer works the first time, or on a second try with gradient PCR, or I redesign it. Of course if I know that there is something special, like high GC or very low GC, I might try DMSO as an additive, or lower the extension temperature. But primer design is, in my experience, the major issue in making PCR work well. Primer3 is your friend.
Phage - I wasn't trying to suggest that every PCR needs to be optimised, just that there are many reasons why it is a good idea. As an aside don't you feel some pride (and suprise) when you have a complex series of experiments all work- I know I do
If you only need a one off PCR and it works under the first condition you try then there is no need to optimise the reaction, however, given the number noobies here I do think it is bad idea advocating not bothering to optimise your PCR reactions.
Oligonucleotide primer walking