Methods for Generating and Analysing
ESTs
In the Filarial Genome Project we use a wide
variety of methods to generate and analyse expressed sequence tags.
Many of these are derived from other workers, and all have been
adapted to improve their efficiency or to suit our needs better. They
are listed here for use by others in the genome project and
elsewhere.
*NOTE* all our protocols are designed to
process clones in a 96 well microtitre plate format. The use of a
multichannel pippette is strongly recommended.
Genome Mapping methods
are listed here
Edinburgh lab protocols for efficient
clone selection, screening and sequencing. *NOTE* all our protocols
are designed to process clones in a 96 well microtitre plate format.
The use of multichannel pippettes is strongly reccommended.
Plating out, picking
and archiving clones from cDNA
libraires.
PCR amplification
of clones and size selection for
sequencing.
Regridding of PCR
products for sequencing size selected clones
SAP/EXO I cleaning of
PCR products and sequencing
Recovery of phage
from DMSO stocks
In order to cross-screen randomly selected cDNA
clones with previously identified "abundant" sequences, we have
developed a relatively low-tech, fast-throughput picking and gridding
scheme, using homemade gridding devices designed to work on 96 well
microtitre plates.
Titreing
libraries
Mass excision of
pBluescript plasmids from lambda Zap phage
Rescuing single
phage stocks
Picking of plaques of
lambda Zap clones into microtitre plates
Picking of colonies
containing excised plasmid cDNA clones into microtitre
plates
Construction of the
multipronged "hedgehog" device for colony
replication
Making replica gridded
filters using a multipronged "hedgehog" device
Regridding of
clones
We have chosen to perform our hybridisation
screening using nonradioactive methods, to make it easier for endemic
country labs to use them.
Making probe DNAs
using PCR from plaques or plasmids
pBluescript sequence map
showing PCR primers
New England Biolabs
"Phototope" Labelling and
detection
Amersham "ECL" Gene
Images labelling
for the record, here is the
Radioactive
Labelling Protocol
