Section II: Troubleshooting a CEQ System Problem
10
CEQ™ 8000 Series Genetic Analysis System
3.
An alternative to preheat treatment is to cleave the plasmid with a restriction
endonuclease before adding the template to the sequencing reaction. In effect,
this method converts a supercoiled plasmid into a linear molecule, which causes
the DNA template to perform like a PCR product (no current problems
observed). This method, although very effective, requires some knowledge of the
DNA sequence to know where to “cut” the plasmid DNA. The plasmid needs to
be cut at a position that is 5' to the sequence that is desired. For example if the
primer binds to the plasmid at position 100 and sequence from position 100 to
600 is desired then the restriction endonuclease recognition site should be at
position 601 or greater.
4.
Removing the template from the sequencing reaction is the only guaranteed
method for curing the current instability problem. This is a very elaborate
process requiring special primers and reagents, but also yields sequencing
reactions that produce no current problems. The most common technique is to
use a biotinylated sequencing primer and the DynaPureII™ sequencing reaction
cleanup kit (Dynal, Inc., Oslo, Norway, product number 603.05). This purifies
the sequencing reaction products from contaminants such as the template DNA,
unincorporated dye-terminators, dNTPs and other salts.
An Identical Erratic current in all eight Capillaries
If all eight capillaries have identical erratic current problems, then the cause of the
problem is a block in the electrical path on the “manifold side” of the CEQ gel system.
A bubble forming in the electrode chamber which blocks the flow of current through
the system most often causes this problem. The result of the blocked current flow is
that all eight capillaries will have reduced read lengths and probable base calling
errors. This is also an indication that there is a system problem and not a problem with
the individual samples. The bubbles may be caused by a variety of reasons: excessive
removal and installation of gel cartridges, improper sealing of the manifold plug of the
capillary array in the gel manifold gasket, a cracked electrode housing, etc.
