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 K177012 operon1_part2

Ania

Tasks:

• Alkaline lysis of the culture containing BBa_B0032 - RBS.3 and BBa_C0012 - lacI repressor on the pSB1A2 ampicillin resistant plasmid.
• Digest of BBa_B0032 - RBS.3 and BBa_C0012 - lacI repressor on the pSB1A2 ampicillin resistant plasmid with XbaI/PstI to obtain the insert
• Digest of 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.

Cloning of the mgtc promoter into the pKSII+ plasmid

Kamil

Tasks:

• Ligation verification
• Bacteria transformation

Methods:

• Correctness of the ligation was verified with gel electrophoresis on 1% agarose gel.

• A 200μl batch of chemocompetent bacteria was transformed with 10μl of ligation mix and incubated overnight on petri dishes containing LB medium supplemented with ampicilin, X-gal and IPTG. (see protocols section for details on transformation).