General PCR Protocol
Sally A. Green, 2-17-96
Here in the Maddock Lab, we do 25μL PCR reactions in 0.5mL microfuge tubes. You can do PCR in different size reaction volumes and in smaller tubes as long as they fit in the thermocycler.
template DNA (genomic, plasmid, cosmid, bacterial/yeast colony, etc.)
primers (resuspended to a known concentration with sterile TE)
buffer (usually 10X, usually sold with Taq polymerase or you can make your own)
note: some different buffer receipes follow at the end of this protocol
MgCl2 (25mM is convenient)
dNTPs (2mM stock)
note: a 2mM stock of dNTPs means that the final concentration of each dNTP (dATP, dCTP, dGTP, and dTTP) is 2mM -- NOT that all dNTPs together make 2mM. dNTPs come as 100mM stocks -- thaw and add 10μL of each dNTP to 460μL of ddH20 to make 2mM. Store at -20°C.
aerosol tips, if desired
PCR is very sensitive to contamination from outside DNAs. Steps should be taken to reduce the chance for contamination, such as wearing gloves, using aerosol tips (tips with a wad of cotton at the top), and not spitting in the tubes. I dont use aerosol tips and have dispensed with the gloves -- just be careful. Something that IS important is to assemble your reactions on ice.
The final concentrations of reagents in PCR reactions are as follows:
buffer: 1X, usually comes as 10X stock. For 25μL reactions, this means 2.5μL.
dNTPs: for most general PCR, you want the final concentration to be 200μM, so a 2mM stock is essentially 10X -- use 2.5μL per reaction.
primers: a good place to start with primer concentration is 50pmol of each primer per reaction. If you dont get your desired product, you can increase to 75pmol or 100pmol. This usually does the trick. I wouldnt go past 200pmol of primers unless its a special protocol that recommends using more.
note: hints on resuspending primers follow this protocol
template: its not usually necessary to be incredibly fastidious about how much template you add to a reaction. You can get product with incredibly small amounts of starting DNA. I usually do a 3mL plasmid prep and use 1/6 of a microliter per PCR reaction. You can use more or less -- it doesnt seem to matter that much.
MgCl2: this is the greatest variable in PCR. The success of a PCR is very dependent on how much magnesium is present in the reaction. For this reason, it is usually advisable to do a magnesium optimization when performing new PCRs. I do my reactions in sets of six, keeping all variables constant except for magnesium. I usually go from 1mM to 6mM MgCl2. Since the stock is 25mM, usually, this means that 1μL of stock equals 1mM MgCl2 in a 25μL reaction -- its convenient.
Given that youre probably going to do at minimum six PCR reactions with six different magnesium concentrations, and you will have to add several different ingredients, its useful to take steps to reduce the number of pipetting steps, which in turn reduces time and change for cross-contamination. The way I set up my reactions is as follows:
I make a chart of how much of each ingredient to add to each tube.