Newcastle University Wetlab/9 September 2008

Tuesday 9th September

 * Half of the overnight cultures iGEMgfp 7.1 and iGEMcherry 13.1 were diluted (by adding 500μl ON culture to 10mL LB) to make the following cultures. These were incubated at 37˚C whilst shaking for 3 hours. 250μl ATCC6633 supernatent was then added to the + ATCC6633 tubes and the mixtures left to incubate for 2 hours at 37˚C.


 * Glycerol stocks made (see protocol) of iGEMgfp 7.1 and 11.1 and iGEMcherry 13.1 and 13.2.


 * Microscopy of the cultures showed similar results to yesterday with again only a possible difference between the iGEMgfp with and without subtilin.


 * BBa_I746107 and pGFPrrnB restricted using 20μl plasmid and 2μl of each enzyme (EcoRI and SpeI) in 100μl total reaction volume. These were run on a gel at 70V for 1 hour.

Lane 1 = 1kb ladder

Lane 2 = pGFP restricted with EcoRI and SpeI

Lane 3 = BBa_I746107 restricted with EcoRI and SpeI


 * This showed successful restriction of both plasmids which were then purified and ligated overnight using 6μl plasmid and 1.5μl enzyme (T4 ligase) in 25μl total reaction volume.