Available Controls, How to Use Them, and What They Tell You
pGEM template + M13F (-20) primer
How to Use Them and What They Tell You:
pGEM template + M13F (-20) primer: First place to start if you’re having problems. This combination run under a variety of different protocols should give clean sequence with a strong signal— at least several hundred “fluorescent units” for each nucleotide. This is a positive control for the PCR machine, enzyme mix, and sample cleanup procedures. The primer and template is provided in premixed format, just add 2 ul (comprising 0.2 ug pGEM and 10 pmol primer) to a standard BigDye reaction, i.e., 2 ul Primer/Template, 3 ul 5X Buffer, 2 ul BigDye, 13 ul water for a total of 20ul reaction.
pGEM template: Add 1 ul to your standard sequence mix with standard primer of your choice (T7, SP6, M13F and R, -20 and –40). This will tell you if those primers are working correctly for you. Also pGem can be used to estimate DNA concentration of your plasmid by running it on an agarose gel along with your sample. DNA is provided at 0.2 ug/ul, presumably quantified correctly by ABI. Spec readings on these preps has given comparable results (between 0.1-0.3 ug/ul).
Amp primer, Km primer: Can be used to test the integrity of your plasmid. Both antibiotic primer sequences are available, depending on the vector you’re using. Provided at a concentration of roughly 10 pmol/ul, use 1 ul. If either of these primers don’t yield good signal, there’s something wrong with your template (providing you’ve run the pGEM template + M13F (-20) primer control with good results).