PCR products
Figure: PCR products
To get good sequencing data from PCR products you must pay particular attention to few things:
1.	First up, optimise you PCR reaction. You must sequence with a single product and a single primer present only. If this is not the case you'll get mixed sequence. If you can't optimise your PCR reaction this far then use a gel purification kit, but you will lose data from this as the DNA template is never as good as from a PCR reaction direct. Of course we can do the cleaning of your PCR products. Then please provide about twice as much DNA as with cleaned fragments, to take some considerable loss of DNA during purification into account.
2.	Next, please clean up your reaction. There are some commercial kits available (Millipore, Qiagen, Macherey & Nagel) to get rid of all your primers, dNTP's and ddNTP's that were in the PCR reaction as they will affect your results. For smaller fragments (less then 300 bp) Qiagen's QIAquick system gives a better yield, for larger fragments we prefer the Millipore Montage or Microcon systems.
3.	Quantify your DNA carefully after purification(cf. section about wrong amount of DNA)!! Remember you are adding the region to be sequenced only there is no extra DNA associated with a PCR product like vector DNA with a cloned fragment. Thus, you can easily add too much template when sequencing directly from a PCR product. We need to have 20 ng/100 bp to conduct our service.
4.	Be aware that there is a limited range in size for PCR fragments that work well in sequencing. Fragments smaller then 150 bp are very difficult to sequence as they work as primers after denaturation and are difficult to purify with good yields. Very large PCR fragments (larger then about 5 kb) are difficult to sequence as well as the probability of mispriming products raises with the length and smaller fragments are amplified more efficiently.
5.	At the end of the PCR fragment the polymerase just drops off the template and the sequence read ends here (cf. figure above). Nevertheless the base calling software tries to interpret the "flatline" and sometimes scales the peaks up for the whole read, thus resulting in a high background noise. At MWG Biotech we avoid this by taking the last part of the run out of consideration automatically. Sometimes a manual clipping and a close look at the chromatogram might give a better sequence.