IGEM:University of East Anglia (UEA), Norwich, UK/2009/Notebook/NRP-UEA-Norwich iGEM/2013/08/21

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Floor One

Made 6 x 500 ml SFM agar, autoclaved for 1 hr and poured into plates (only 3 x 500 ml had 500 ul Nystatin added).

Performed 24 spore stocks of lawns - filtered, concentrated and resuspended spores in 20 % glycerol.

Looked at spore titres. A total of 13 of the 15 spore stocks titred had the a good number for succesful Bioassay of 10^-8 spores, although lower numbers would still work.

Floor Two

Minipreps of BL21 containing pETAntA and AntGp+RFP plasmids were prepared using 5ml from each overnight culture and incubated with PstI restriction enzyme for analysis by gel electrophoresis fig 2. We also prepared the biobrick BBa_K1041000 (sample 3b2) by cutting with restriction enzyme NdeI and analysing on an agarose gel fig 2. The large fragment was cut from the gel to be purified. This process should remove the extra NdeI sites that were introduced to the sequence.

Fig 2: Analysis of Restriction enzyme digests with PstI and NdeI. Lane 1- 4 contain samples 1-4 of BL21(pETAntA +AntGpRFP) digested with PstI. Lane 6 and 7 contain uncut pETAntA and AntGpRFP plasmids respectively. Lane 9 contains sample 3b2 digested with NdeI. The larger band of lane 9 was excised from the gel.

100μL from two of the overnight cultures was used to inoculate two fresh 10ml LB with added antibiotics Kanamycin and Chlorophenicol. They were incubated for 2 hours at 37°C and then one culture was induced with IPTG. Both cultures were left in a shaking incubator at 25°C overnight.


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