IGEM:University of East Anglia (UEA), Norwich, UK/2009/Notebook/NRP-UEA-Norwich iGEM/2013/08/22
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Diluted soil samples 79-84 from 10^-1 to 10^-4 and plated 10^-2 to 10^-4 on SFM + Ny.
Set up more Bioassays from spore stocks - spotted 2 x 5 ul of spore stock onto a single SFM plate. The plates were left to grow at 30 ° for 7 days.
Set up overnight ETpuz cultures for tomorrow's conjugation. Added 50 ul E. coli cells to ~ 10 ml LB (lacking NaCl for ETpuz pMS82 cells as Hygromycin is salt-intolerant). Added 10 ul Chloramphenicol and Kanamycin to both ETpuz pAU3-45 and pMS82 cultures, and 10 ul of Apr for pAU3-45 and 10 ul of Hyg for pMS82.
We discovered after analysing the sequencing data from 18/07 that sample 3a had the correct sequence for biobrick BBa_K0141000 but sample 3b did not. This explained why sample 3b2, which was a miniprep from 3b, had extra NdeI sites. To confirm our theory, samples 3a, 3b, 3c and the original BBa_J04450 were cut with restriction enzyme pvuII and analysed by gel electrophoresis fig 3. The results show that sample 3b does appear to have extra DNA as the lowest band is largest than the ones for 3b and cut BBa_J04450. So the excised gel from the previous day was ignored. Although colonies were visible on the LB plates (BL21 cells containing pETAntA and antGpRFP plasmids) they do not appear red. The plates were left to incubate longer at 37°C and the cells were harvested from the two cultures.
The registration for the jamboree was completed for the team.