Template Tips
The types of DNA template which can be sequenced are double
and single-stranded plasmids, PCR products, cosmids and artificial
chromosomes. Regardless of template type, it is important to have a
good quality template that is:
- Free of contaminating salts, solvents, RNA, proteins and
chelating agents
- Homogeneous
With quality template we routinely achieve read lengths
of greater than 700 bp. Good sequencing results are obtained from
template purified using commercially available kits or carefully
executed alkaline lysis and PEG precipitation protocols. We have
available the recommended procedures for preparation of templates. If
you should require these protocols, please do not hesitate to contact
us.
Concentrations measured using standard spectrophometric
techniques will only be accurate for highly pure DNA. Please only use a
spectrophotometer reading where the A260/A280 ratio is >1.8.
Impurities such as proteins, oligonucleotides, and unincorporated
nucleotides prevent accurate spectrophometric measurements and the
actual yield can be significantly lower than the concentration
indicated.
For double-stranded DNA, ethidium bromide fluorescence on an
agarose gel may be inaccurate as its fluorescence is not specific to
double-stranded DNA. A mass ladder should be used for comparison in
this method.
We quantitate DNA using H-33258 fluorescence enhancement,
which is highly sensitive, specific and accurate. Whilst this method
provides accurate quantitation of DNA even in the presence of RNA,
protein, nucleotide, dilute detergents and protein denaturants, the
presence of any of these factors will adversely affect the sequencing
reaction.
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