Team:Warsaw/Calendar-Main/21 July 2009
From 2009.igem.org
Transformation of E. coli with pKS/hly plasmid
Kama
- chemocompetent E. coli dH5α were incubated on ice for 15 minutes
- 15μl of ligation mixture was added (and 15μl of control ligation, without insert)
- bacteria were incubated with DNA on ice for 30 minutes
- heat shock was conducted (1 minute 42°C)
- bacteria were incubated on ice for 3 minutes
- After the heat shock 800μl of SOB medium was added
- Mixture with bacteria was incubated for 1 hour in 37°C
- 100μl of mixture was plated on medium containing ampicillin, X-Gal and IPTG
- The rest of mixture was rotated for 1 minute, pelet was
Construction of [http://partsregistry.org/wiki/index.php?title=Part:BBa_K177012 K177012] operon1_part2
Ania
Tasks:
- Alkaline lysis of the culture containing [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0032 BBa_B0032 - RBS.3] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0012 BBa_C0012 - lacI repressor] on the pSB1A2 ampicillin resistant plasmid.
- Digest of [http://partsregistry.org/wiki/index.php?title=Part:BBa_B0032 BBa_B0032 - RBS.3] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0012 BBa_C0012 - lacI repressor] on the pSB1A2 ampicillin resistant plasmid with XbaI/PstI to obtain the insert
- Digest of [http://partsregistry.org/wiki/index.php?title=Part:BBa_C0012 BBa_R0051 - cI regulated Promoter] on the pSB1A2 ampicillin resistant plasmid with SpeI/PstI to obtain the insert
- Gel
- DNA samples were cut out and frozen in the seperate eppendorfs (still within the agarose gel).
[image:] Results:
[image:]
Comments:
- There is a vector with PcI clearly visible in the first two samples. The PcI is too short to be visible on its own. The insert, that is RBS.3+LacI is visible as the 1200 band in the last three samples. The vector from two first and the insert from two last samples were cut out of the gel.
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