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Team:BIOTEC Dresden/Notebook3-1
From 2009.igem.org
(New page: =='''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 tomorro...) |
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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. | 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. | ||
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Red/ET for insertion of FRT-GFP-BsdR into the Fosmid-SpecR: Cultures were set up. | Red/ET for insertion of FRT-GFP-BsdR into the Fosmid-SpecR: Cultures were set up. | ||
| - | + | '''2nd October''' | |
Red/ET recombination was done for: | Red/ET recombination was done for: | ||
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1. pTetFlp-KanR-CmR + F3-ZeoR-F3-RFP | 1. pTetFlp-KanR-CmR + F3-ZeoR-F3-RFP | ||
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2. Fosmid-SpecR +FRT-GFP-BsdR | 2. Fosmid-SpecR +FRT-GFP-BsdR | ||
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All plates incubated at 30° C o/n | 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 GB2005-DIR and GB2005-Red cells were plated on LB and kept at 37° C o/n. | ||
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The #1 to#24 (from 1st oct) were digested as follows: | The #1 to#24 (from 1st oct) were digested as follows: | ||
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sample #1 and #2 (F3-Zeo-F3-RFP): by XhoI | sample #1 and #2 (F3-Zeo-F3-RFP): by XhoI | ||
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sample #3 to #8 (FRT-GFP-BsdR): by HincII | sample #3 to #8 (FRT-GFP-BsdR): by HincII | ||
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sample #9 to #17 (pMA-FRT-dhfr) and #18 to #24 (pRha-Flp-CmRKanR): by PvuII | 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 plates in the 37°C, incubator (GB2005-Red & GB2005-DIR) were kept in the fridge. | ||
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-Fosmid-Spec+FRT-GFP-BsdR | -Fosmid-Spec+FRT-GFP-BsdR | ||
did not have any colonies, so they were kept @30° C for another day. | did not have any colonies, so they were kept @30° C for another day. | ||
| + | {{:Team:BIOTEC_Dresden/NewTemplateEnd}} | ||
Revision as of 11:09, 21 October 2009
News
Finally, the Gene Expression Kit arrived. Logo design progresses.
Sponsors
[http://www.biotec.tu-dresden.de 
]
[http://www.nexans.it 
]
[http://www.sud-chemie.com 
]
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.
