---+ Keio and ASKA collections The [[http://www.shigen.nig.ac.jp/ecoli/strain/top/top.jsp][Keio and ASKA collections]] are very useful resources for _E. coli_ genetics. The *Keio collection* consists of single-gene knockout strains in the K12 BW25113 strain background (an MG1655 derivative). They contain a Kan^R resistance cassette in place of the gene. One can easily remove this cassette to create an in-frame deletion of the gene using FLP recombinase expression from plasmid pCP20. The *ASKA collection* is a set of ORFs cloned from _E. coli_ K12 W3110 into the high copy number plasmid pCA24N. This plasmid has a modified pMB1 replication origin (same as pQE30 from Qiagen) and Cam^R marker. The cloned ORF is under control of the IPTG-inducible T5-lac promoter. Our samples of these strain collections were transferred from 96-well microplates into 384-well microplates using a 96-well pin tool. Thus, the plates are interspersed with one another, with one plate in the upper-left register, the next plate in the upper right register, the third plate in the lower left register, and the final plate in the lower right register. This is the layout in the order listed on the plate label. The attached spreadsheets can be used to determine if a strain exists in the collection (be sure you search gene synonyms) and to find the well number for a desired strain to retrieve it. *Important!* When retrieving a strain from the freezer and regrowing it: 1 Streak out to a single colony on an LB agar plate with the correct antibiotic. 2 Verify that the strain/plasmid is correct. * By comparing PCR product size or a known phenotype to BW25113 for Keio Strains [[http://barricklab.org/twiki/bin/view/Lab/ProtocolsFreezingStrains][PCR Protocol]] * By sequencing the plasmid insert for ASKA strains. Sequencing primers [[http://barricklab.org/adminer/index.php?username=lab&select=primers&where%5B0%5D%5Bcol%5D=&where%5B0%5D%5Bop%5D=&where%5B0%5D%5Bval%5D=SPL&where%5B1%5D%5Bcol%5D=number&where%5B1%5D%5Bop%5D=%3C%3D&where%5B1%5D%5Bval%5D=4&where%5B2%5D%5Bcol%5D=number&where%5B2%5D%5Bop%5D=%3E%3D&where%5B2%5D%5Bval%5D=3&where%5B3%5D%5Bcol%5D=&where%5B3%5D%5Bop%5D=&where%5B3%5D%5Bval%5D=&limit=30][SPL003 and SPL004]] (requires login) flank the ORF insertion site in the pCA24N and are available in the Welch lab. [[http://barricklab.org/twiki/bin/view/Lab/ProcedureDNASequencing][DNA Sequencing Protocol]] 3 Create a stock of this verified isolate in a cryovial for further use by the lab.

   

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Topic attachments

I	Attachment	History	Action	Size	Date	Who	Comment
xls	Platesheet__ASKA_96_384.xls	r1	manage	368.5 K	2014-06-26 - 22:07	SeanLeonard	Spreadsheet updated to include clone locations on 384 well plates
xls	keio.collectionsheet.xls	r4 r3 r2 r1	manage	856.0 K	2014-05-30 - 10:56	JeffreyBarrick	Keio collection microplate layout, including 96- and 384-well plate versions

 Barrick Lab  >  ProtocolList  >  ReferenceKeioASKACollection

Contributors to this topic JeffreyBarrick, SeanLeonard, CraigBarnhart, SimonDAlton, DanielDeatherage

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