Team:UIUC-Illinois/Notebook

1. Prepare electrocompetent cells that have been transformed with the helper plasmid (pAH57) and that have been grown in 5 mL SOB to an OD of 600nm of ca. 0.6 with ampicillin antibiotic at 30 degC.

2. Perform an electroporation transformation with the BioBrick Compatible Lambda Chromosomal Insertion Plasmid making sure to resuspend the cells in 1 mL of SOC (without ampicillin). Incubate at 37 degC for 1 hour, and then 42 degC for 30 minutes. Spread plate onto a selective agar plate and incubate at 37 degC.

Day 1

The CRIM pAH125 vector was obtained in a pir+ strain. The culture was streaked from the -80C onto a LB kanamycin 30ug/mL plate. Plate was incubated 37C for 18 hours. White colonies resulted.

A single colony was picked from the previous plate and was used to inoculate 6mL of LB kanamycin 30ug/mL. The culture was then incubated 37C on a tube turner for 16 hours. A turbid yellow-white culture resulted.

5mL of the culture was pelleted and miniprepped using the Promega mini-prep kit and protocol described in our protocol download. The resulting concentration was 30ng/uL.

The following primers were then designed in order to amplify the highlighted (red)section below. The primers contain overhangs such that subsequent SpeI digestion and an intramolecular ligation of the pcr product will produce a biobrick cloning site, which replaces the original MCS and lacZ ORF in the pAH125 diagram.