GE 33 Recording Equipment User Manual


 
12
Preparation of template DNA
Since cycle sequencing can be performed using very little template DNA, only
very small amounts of detrimental impurities are likely to be carried along with
the DNA. Therefore, though still important, template purity may not be as crucial
for cycle sequencing as it is for non-cycle sequencing.
Preparation of single-stranded template DNA
Single-stranded template DNA of good purity is essential for excellent
sequencing results. Several popular plasmid cloning vectors contain the same
lac-derived cloning region as the M13mp vectors and a single-stranded phage
replication origin. Production of single-stranded DNA from these vectors is
similar to that of the M13 phage and the single-stranded DNA produced can
also be used as template for sequencing.
Preparation of double-stranded plasmid DNA
Sequencing double-stranded templates with the Thermo Sequenase
Radiolabeled Terminator Cycle Sequencing Kit works effectively with no
changes in the reaction protocol. Alkaline denaturation is not required for
plasmid DNA templates. For best results, purified plasmid DNA should be
used—CsCl gradients, PEG precipitation, adsorption to glass, columns, and
other common DNA purification methods all produce suitable DNA. (However,
since such small quantities of DNA are added to the reactions, even impure
DNA samples can sometimes yield acceptable sequence data.) There are many
popular protocols for purifying plasmid DNA from 2-10ml cultures. We have had
consistent success with ‘boiling’ (21) and ‘alkaline’ (22) mini-prep methods.
Cycle conditions and template quantity
The temperatures used for cycling the termination reactions should be
determined from the characteristics of the sequencing primer, the template, and
the length of the termination product desired. The number of cycles required will
depend on the quantity and quality of the template DNA used. The following
guidelines should assist in choosing cycling parameters.
Cycling temperatures
The melting temperature of the primer should be kept in mind when choosing
cycle temperatures. The control primer included in the kit is moderately long (23
bases) with 50% G/C content. The melting temperature of this primer is ~73°C
under sequencing reaction conditions, and excellent results are achieved by
cycling between 60°C and 95°C. The duration of the steps does not seem to be
critical, and even brief pauses (1-10 seconds) at these temperatures seem to
be effective (except with dITP as described above).