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Uppsala-Sweden/12 August 2009
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| + | ==Evaluation of competent cells, continued== | ||
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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. | ||
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| + | ==Colony PCR, continued== | ||
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| + | The PCR from yesterday was evaluated on a gel, and the result is as shown bellow. | ||
| + | [[Image:Example.jpg]] | ||
| + | |||
| + | ==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 | ||
| + | --[[User:Flormane|Flormane]] 14:59, 12 August 2009 (UTC) | ||
Revision as of 14:59, 12 August 2009
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.
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)
