Knock- Out Cassettes for A. nidulans
List of cassettes and primers *
Kinase
strain list
Kinase set
in plates from the FGSC
(fees)
Gel images are available for the cassette plates
These cassettes were produced at Dartmouth Medical
School and have been shipped to the FGSC as DNA suitable for
transformation or amplification.
We received two sets of 96 well plates.
The first holds 60 ul of cassette DNA per well and the second holds
mixed primers for reamplification of the cassette.
The primers are in the
same locations as the cassettes and are at a working concentration of
10um.
Please request cassettes by plate number and location.
Dartmouth has sent recommended parameters for re-amplification of cassettes:
Dilute the cassette template at least
100-fold and use the following cycling parameters:
1
35 cycles
10 min 72 C
These parameters are for
Requests for cassettes should be limited to those that will be used to generate knockout mutants immediately. Strains should be deposited in the FGSC as soon as practical.
The FGSC will ask $2 per cassette or primer pool.
The selectable marker is A. fumigatus pyrG.
*Most of the fields in the spreadsheet are self explanatory. The
columns "cassette" and "flanks" contain an entry if there were failures
generating the cassette. The possibilities are flank failures or PCR
failures.
The column "OCC" indicates that a gene is represented twice.
This is important to note because there were large numbers of failures
on certain plates. Following an initial failure, new primers were
ordered and the PCR was repeated followed by transformations which then
succeeded. The column "Exclude" indicates which of these genes failed
the first time because of primers.
A small group of A. niger
cassettes were produced and the yeast genomic DNA for final
amplification of the cassettes was sent to the FGSC.
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9/22/11