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Imperial College/4 September 2008
From 2008.igem.org
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=4 September 2008= | =4 September 2008= | ||
==Wetlab== | ==Wetlab== | ||
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| + | ===Cloning=== | ||
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| + | *Pfu PCR reactions for LacI gene, Aad9 gene and AmyE 3' Integration Sequence set up using the standard conditions and reagents [http://openwetware.org/wiki/IGEM:IMPERIAL/2008/Prototype/Wetlab/PCR]; LacI and AmyE 3' at 52°C for the first 10 cycles and 65°C for the last 20, Aad9 at 52°C then 60°C. 5μl of each PCR reaction run on a gel - results below | ||
| + | *CAT biobrick digested with ''EcoRI'' and ''SpeI'' ready for assembly with the terminator | ||
| + | *Termiantor biobrick cut with ''EcoRI'' and ''XbaI'' ready for assembly with CAT | ||
| + | *GFP-Termiantor biobrick cut again with ''XbaI'' and ''SpeI'' to produce biobrick vectors for our PCR clones | ||
| + | *GFP-Termiantor biobrick cut again with ''EcoRI'' and ''SpeI'' to produce biobrick vectors for our GeneArt produced clones | ||
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===Preparation of Electrocompetent ''E. coli'' Cells=== | ===Preparation of Electrocompetent ''E. coli'' Cells=== | ||
We have prepared 42 aliquots of electrocompetent XL1 Blue ''E. coli'' cells using [http://openwetware.org/wiki/IGEM:IMPERIAL/2008/New/Protocols/XL1-Blue_preparation the relevant protocol] listed on our Protocols Page. The protocol was updated to include a higher innoculation value from the overnight culture. | We have prepared 42 aliquots of electrocompetent XL1 Blue ''E. coli'' cells using [http://openwetware.org/wiki/IGEM:IMPERIAL/2008/New/Protocols/XL1-Blue_preparation the relevant protocol] listed on our Protocols Page. The protocol was updated to include a higher innoculation value from the overnight culture. | ||
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| + | ===Results=== | ||
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| + | [[Image:4-08PCR.PNG|thumb|600px|center|A 1% Agarose gel showing the results of various PCR reactions. Each lane is loaded with 5ul of PCR reaction and 1ul of 6x sample buffer. M = Marker, lane 1 is the AmyE 3' Integration sequence, lane 2 the LacI and lane 3 the Aad9 PCR reaction]] | ||
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| + | As can be seen, both the AmyE 3' integration sequence and the Aad9 (spectinomycin resistance) gene amplified properly. LacI did not however and so this will need to be repeated. | ||
Revision as of 17:37, 5 September 2008
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4 September 2008WetlabCloning
Preparation of Electrocompetent E. coli CellsWe have prepared 42 aliquots of electrocompetent XL1 Blue E. coli cells using [http://openwetware.org/wiki/IGEM:IMPERIAL/2008/New/Protocols/XL1-Blue_preparation the relevant protocol] listed on our Protocols Page. The protocol was updated to include a higher innoculation value from the overnight culture. Results |
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