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).

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.