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NR-19437   Aspergillus fumigatus, Knockout Gateway® Clone Set, Recombinant in Escherichia coli
(Clones)
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at no cost to registered researchers.
Description: Aspergillus fumigatus, Knockout Gateway® Clone Set, Recombinant in Escherichia coli
Organism: Aspergillus fumigatus
Biosafety Level: 1
Availability Status: In Stock
Store at: -80°C or colder
Contributor: Pathogen Functional Genomics Resource Center at the J. Craig Venter Institute
Comments:
Quantity limit per order for this item is 1. This item can be ordered twice a year. Orders over this limit will be sent to NIAID for approval before shipment.

The Aspergillus fumigatus (A. fumigatus), Gateway® clone set consists of 1 plate which contains 70 sequence validated knockout clones from A. fumigatus, strain A1163 with ampicillin selectable pyrG selection gene, cloned in Escherichia coli (E. coli) DH10B-T1 cells. Each open reading frame was constructed in vector pDEST™ 17 (Invitrogen™). Successful cloning was determined using a restriction digestion approach.

Information related to the use of Gateway® Clones can be obtained from Invitrogen™. Using the three fragment MultiSite Gateway® strategy the pyrG selectable marker was assembled with chromosomal amplicons (~ 1Kb) of the regions flanking the targeted gene. The three fragments (left flank, pyrG, right flank) were amplified and cloned into entry vector pDONR™221 P1-P4, pDONR™221 P4r-P3r and pDONR™221 P3-P2, respectively. The flanking clones were confirmed by PCR, the pyrG clone was sequence verified. The final assembled construct is created using LR Clonase™ II Plus to recombine the three clones with each other and the destination vector, pET-DEST-TIGR02. Linearized vector can be used to transform chemically competent protoplasts. Upon homologous recombination the pyrG gene complements an auxotrophic mutant A1163 strain. Please refer to the Invitrogen™ Gateway® Technology Manual for additional details.

Plate orientation and viability were confirmed for NR-19437.

Each inoculated well of the 96-well plate contains approximately 60 µL of E. coli culture (strain DH10B-T1) in Luria Bertani (LB) broth containing 100 µg/mL ampicilln supplemented with 15% glycerol.

Clone plates are replicated using a BioMek® FX robot. Production in the 96-well format has increased risk of cross-contamination between adjacent wells. Individual clones should be purified (e.g. single colony isolation and purification using good microbiological practices) and sequence-verified prior to use. BEI Resources only confirms the clone plate orientation and viability of randomly picked clones. BEI Resources does not confirm or validate individual clone identities provided by the contributor.
Citations: Acknowledgment for publications should read "The following reagent was obtained through BEI Resources, NIAID, NIH: Aspergillus fumigatus, Knockout Gateway® Clone Set, Recombinant in Escherichia coli, NR-19437."
Additional Information: Limited Use Label License
NR-19437 JCVI Plate Map
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  • Customers located in the state of Hawaii will need to contact the Hawaii Department of Agriculture to determine if an Import Permit is required. A copy of the permit or documentation that a permit is not required must be sent to ATCC in advance of shipment.

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