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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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| + | '''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. | 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}} | ||
Latest revision as of 11:58, 21 October 2009
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.
