Plant Molecular Biology Center
Core DNA Analysis Lab
Greetings from the core. This lab is equipped with a Beckman CEQ8000XL capillary system for DNA sequencing and fragment analysis. We also have a Stratagene Mx3000P system for quantitative real-time PCR. Users of this facility are primarily from N.I.U. but we can accept sequencing requests from others. Primers used for sequencing should have a Tm of at least 55 C and have a G or C at the 3' end. It is also good to check for potential secondary structure and self-dimer formation. This can be checked on the web using the oligo
analyzer. Degenerate primers (those having a mixture of bases at certain positions) may not work for sequencing. Another important consideration for successful DNA
sequencing is that template DNA must be pure. I have had good results with this mini scale preparation of plasmid DNA
for fluorescent sequencing.
Some commercial plasmid DNA
purification kits, such as the Wizard Plus SV miniprep from Promega and the Nucleospin Plasmid prep from Macherey-Nagel yield good template. An additional sodium acetate/ethanol precipitation followed by a 70% ethanol wash of plasmid DNA may improve sequence quality. Problems may arise if phenol is used in any of the DNA
purification steps. Impurities from the phenol can poison the polymerase used
in the sequencing reaction. It is always useful to run template DNA on an agarose
gel before use in sequencing. The gel will aid in DNA quantitation, and help one to determine if the DNA is
undegraded, and if RNA is present in the sample.
In addition to plasmid DNA, PCR products can also be used as templates for
sequencing. It is good to keep in mind that the standard annealing temperature that I use for cycle sequencing is 50 C. When the annealing temperature for PCR is less than 50 C, products from these reactions may not be suitable templates for cycle sequencing under standard conditions. If a single PCR product is seen after electrophoresis, a PCR
can be treated to degrade or remove oligos and dNTPs prior to sequencing. I have had good
results with the Amersham PCR product pre-sequencing kit. For purification of larger PCR volumes I prefer the quickstep 2 PCR purification kit from EdgeBiosystems. If more than one band
(product) is present in a PCR, it may be necessary to purify the band of interest
from a gel. A cheap method is listed below. If you prefer to buy a kit, I
recommend use of the Qiagen QIAquick gel extraction kit or the Amresco cyclopure gel extraction kit.
Some handy links follow:
