Sample requirements
Host strains
These
differ in growth characteristics, plasmid copy number, endonuclease
expression and other contaminants such as carbohydrates and
glycoproteins that can inhibit sequencing polymerase
expression. DH5-alpha, HB101, XL-1 Blue are consistently good.
JM109 and MV1190 are usually fine but JM101 is often poor. The
growth media you use can also affect yields, LB is usually
fine.
Template preparation
DNA that
is to be sequenced must be free from other template, for example,
mixed clones or PCR products, genomic DNA and RNA. In addition,
your samples should be free of anything that may inhibit the Taq,
for example, EDTA and salt.
Plasmid Preparation
Alkaline
lysis with RNAse and PEG precipitation can give very good
results. There are many commercial kits available. These
are usually either Ion-exchange resin based or silicon based.
Ion-exchange columns usually give very good results but care must
be taken not to overload the columns. The DNA is eluted in high
salt so you should always perform a desalting step, for example, a
precipitation with 70%EtOH wash or use a spin column.
Silica
based kits are usually inexpensive but can also give good results
but care must be taken to achieve the best results. When the DNA is
eluted in water salt is often eluted as well so it is advisable to
add an extra desalting step.
Care also must be taken to avoid any silica fines in the eluted
DNA (the silica fines can bleed through the columns and cause death
to enzymes.) This can be done by performing a long spin on the
eluted DNA and removing (and keeping!) the supernatant.
Details of our
recommended kits that we use in house
Cosmids, BACs/P1's
Alkaline
lysis with PEG precipitation give good results.
PCR Products
We request
that you fully optimised the PCR which would reduce the amount of
clean up required.
The simplest form of clean-up is simply to dilute an aliquot of
the PCR reaction between 1:5 and 1:10 in water. Please ensure that
the final concentration of the PCR primers is less than 0.2uM and
the dNTP's is less than 100uM. This method can give good results on
highly optimized PCR's.
This
method can give good results on highly optimized PCR's.
Another method is using Exonuclease I/ Shrimp Alkaline Phosphatase.
The exonuclease degrades the left over PCR primers and the alkaline
phosphatase dephosphorylates the dNTP's so they won't interfere
with the sequencing reaction (they tend to out-compete the
flourescently labelled dNTP's). This method does not, however,
remove any secondary products of the PCR which can lower the
quality of the data. In order to get rid of small DNA fragments, we
suggest column purification or gel purification. With column
purification, these can be silica based, gel filtration or ultra
filtration and will isolate DNA above a particular size (eg
120bp).
With gel purification, the best method is for
non-optimised reactions as it isolates the fragment you want from
secondary products, primers and nucleotides. This method can be
time consuming and the visualisation of the gel with UV light can
cause "nicking" of the DNA.
Details of our recommended kits that we use in
house
Template Quantitation
Sequencers are able to handle a wide range of DNA concentrations
however with very low amounts of DNA the data quality will be
significantly affected. Using UV absorbance to measure the quantity
of DNA in dilute DNA solutions tends to give widely inaccurate
results. A good way to measure the quantity of DNA is to run
an aliquot on a mini-gel and compare the intensity to a control of
known concentration. There are also concentration ladders that are
commercially available.
Sample Storage
We guarantee to keep your templates and primers up-to 3 weeks,
unless instructed otherwise. If you would like any of your
materials kept for longer, then simply let us know and we will move
them into longer term storage. Materials in long term storage that
have not been used for a period of 3 months will be disposed of,
unless you instruct us otherwise.
If you would like to discuss any of these points further, please
do not hesitate to contact us.