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Team:BIOTEC Dresden/Notebook3-1
From 2009.igem.org
1st October
The concentration of the previous minis was very low, so mini prep was repeated. Again measured concentration were found to be right. These would be digested tomorrow.
Red/ET for insertion of F3-Zeo-F3-RFP into pTet-Flp-KanR and pRhaFlp-CmR-KanR:
-overnight cultures were set up by picking 2 colonies from the GB2005-DIR plate. -These colonies were inoculated into 2 tubes containing 1.4 ml LB each kept o/n @ 37°, 1000 rpm.
Red/ET for insertion of FRT-GFP-BsdR into the Fosmid-SpecR: Cultures were set up.
2nd October
Red/ET recombination was done for: 1. pTetFlp-KanR-CmR + F3-ZeoR-F3-RFP 2. Fosmid-SpecR +FRT-GFP-BsdR
These were induced using L-Arabinose. The samples are plated as following:
-pTetFlp-KanR-CmR + F3-ZeoR-F3-RFP Kan plate and kan + Zeo plate (in duplicate) -pTetFlpKanR - no insert kan, Zeo+kan -Fosmid-SpecR+FRT-GFP-BsdR Bsd, Bsd+Spec -Fosmid-SpecR-no insert Bsd, Bsd+Spec
All plates incubated at 30° C o/n The GB2005-DIR and GB2005-Red cells were plated on LB and kept at 37° C o/n. The #1 to#24 (from 1st oct) were digested as follows: sample #1 and #2 (F3-Zeo-F3-RFP): by XhoI sample #3 to #8 (FRT-GFP-BsdR): by HincII sample #9 to #17 (pMA-FRT-dhfr) and #18 to #24 (pRha-Flp-CmRKanR): by PvuII
3rd October
The plates in the 37°C, incubator (GB2005-Red & GB2005-DIR) were kept in the fridge.
The other plates
-pTetFlp-kanR+F3-Zeo-F3 RFP
-Fosmid-Spec+FRT-GFP-BsdR
did not have any colonies, so they were kept @30° C for another day.
