Violetearth

If you are just testing PCR capacity, perhaps you could get a plasmid that comes with standard primers and run it with your protocol. Someone in another lab would likely loan you the minute amounts you would need. In fact, if you tell them what you are doing, they might give you primers and DNA that they know will work. In response to your thoughts: 1. The DNA extraction was unsuccessful. - You can test your extraction by quantifying the resulting DNA at 260 nm on a spectrometer, or - You can cut some DNA with a high frequency cutting plasmid and run the DNA on a gel next to a marker that allows you to quantify your DNA yield. This is a bit barbaric, but effective for a yes/no answer. 2.The primer. I agree with the previous post. This may be the first thing to check. If your sequence does not match anything in the genome or if it only primes in one direction, you will not get amplification. You should find two primer-appropriate genomic sequences that flank a gene of interest. One should run 3' to 5' and other should run 5' to 3' so that bases are added across the gene of interest. 3.Water not TE. Sterile, de-ionized water works beautifully for resuspending primers. This is not a concern. 4.Hot start Hot start means that instead of the standard 2 - 3 minute denaturing temperature, your first PCR cycle has a 10ish minute denaturing cycle. This is bad for your regular Taq. You can manually hot start by adding your Taq to the reaction at the 7 minute stage so it is only in the heat 2 - 3 minutes. This can greatly increase the amount of product you get from your reaction. 5.Reduce the annealing temp Probably not. Your primer is GC rich and should anneal if it has a mate in the genome. 6.Decrease or increase the denaturing temp. Increase it if the genome of your species is extremely GC rich. Otherwise, you're probably okay.

You are right to think that proteins and ATP are often used by the cell that makes them. In a multi-cellular organism, they may also act in the extracellular environment or in other cells. If you think about it, unicellular organisms may also produce proteins that are excreted into the environment where they may interact with the cells of other organisms.