Knock- Out Cassettes for A. nidulans


    Strategy document

    List of cassettes and primers *

    Kinase strain list   

    Kinase set in plates from the FGSC

    Selectable marker sequence

    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: 
min 94 C

30 sec 94 C
30 sec 60 C
5 min 72 C
        35 cycles

    10  min 72 C
    These parameters are for
LA Taq in a 50ul reaction, 1 ul of the 5f/3r primer mix (.2uM final each primer) and 400uM each dNTP.

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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