Sequencing phage and cosmid DNA can be a little more difficult. The keys to good sequencing of phage and cosmid DNA seem to be quality and quantity. Be sure that you have a very clean prep.
Phages and cosmids must be brought to us in water at 0.2µg/µl concentration. We need 1.0µg of plasmid per reaction. The correct quantitation is very critical with these larger templates. More is not better, in fact, if any rule is true, it is that less is better. For this reason we recommend that you take added precautions when quantitating your DNA. First, be sure to allow the template to resuspend at least overnight (on the bench or in the refrigerator) before attempting to quantitate. This insures that the template concentration will not change after quantitation. NEVER freeze these large templates, as they will come out of solution. They will be just fine in the refrigerator. Secondly, determine concentration by O.D. and confirm that reading by running on a gel. DO NOT bring your samples in TE. EDTA chelates magnesium, and Taq has an absolute requirement for magnesium, so the reactions do not work if the samples are in TE.
Primers should be in water at 1µM concentration. Please do these calculations and make the dilution yourself. If you have a primer synthesized in our facility that will be used for sequencing, you may check the "Please give oligo to DAF for sequencing" box on the oligo request form.