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Uppsala-Sweden/12 August 2009
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The competence of the cells were evaluated as given by the Invitrogen protocol. The colony number at 500 pg of pUC19 plasmid and 500 µl of end culture gives an transformation factor of 7,2*10^6, which is good. | The competence of the cells were evaluated as given by the Invitrogen protocol. The colony number at 500 pg of pUC19 plasmid and 500 µl of end culture gives an transformation factor of 7,2*10^6, which is good. | ||
| + | edit 7,2*10^6 is actually quite crappy compared to what you're expected <br> to get considering the top ten protocol form open wetware. <br> There it says that good cells should <br> yield 500 - 5000 *10^6 cfu/µg pUC19. So, conclusion, <br> Invitrogen makes crappy cells ;) | ||
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| + | --[[User:Anders|Anders]] 15:28, 13 August 2009 (UTC) | ||
==Colony PCR, continued== | ==Colony PCR, continued== | ||
The PCR from yesterday was evaluated on a gel, and the result is as shown bellow. | The PCR from yesterday was evaluated on a gel, and the result is as shown bellow. | ||
| - | [[Image: | + | [[Image:20090811-colonypcr1-labelled.jpg]] |
==Transformation of ADH2 from z.mobilis== | ==Transformation of ADH2 from z.mobilis== | ||
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The final DNA conc was ~34 µg/ml | The final DNA conc was ~34 µg/ml | ||
--[[User:Flormane|Flormane]] 14:59, 12 August 2009 (UTC) | --[[User:Flormane|Flormane]] 14:59, 12 August 2009 (UTC) | ||
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| + | ==kivd design== | ||
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| + | I codon-optimized the kivd gene using this codon-usage table (synechoystis and E. coli optimuzed) | ||
| + | [[Media:codon_usage.xml]] | ||
Latest revision as of 15:38, 16 October 2009
Contents |
Evaluation of competent cells, continued
The competence of the cells were evaluated as given by the Invitrogen protocol. The colony number at 500 pg of pUC19 plasmid and 500 µl of end culture gives an transformation factor of 7,2*10^6, which is good.
edit 7,2*10^6 is actually quite crappy compared to what you're expected
to get considering the top ten protocol form open wetware.
There it says that good cells should
yield 500 - 5000 *10^6 cfu/µg pUC19. So, conclusion,
Invitrogen makes crappy cells ;)
--Anders 15:28, 13 August 2009 (UTC)
Colony PCR, continued
The PCR from yesterday was evaluated on a gel, and the result is as shown bellow.
Transformation of ADH2 from z.mobilis
This time we tried a new approach and did the digestion on a column instead, along with a normal approach as control. For the digestion on a column we put 10µl of 4ng DNA (PCR product) and ran it through a Invitrogen PCR clean up column, without final elution. To the membrane we added 20µl of Fermentas digestion buffer (mixed as given by protocol) and added (2+2) µl of digestion enzyme (EcoR1 and PstI) this wa incubated in 37 deg. celsius for 30 min. After this the reaction was aborted by a second Nucleospin PCR clean up on the same column. The final DNA conc was ~34 µg/ml --Flormane 14:59, 12 August 2009 (UTC)
kivd design
I codon-optimized the kivd gene using this codon-usage table (synechoystis and E. coli optimuzed) Media:codon_usage.xml
