Team:BIOTEC Dresden/Notebook3-1

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.