IGEM:University of East Anglia (UEA), Norwich, UK/2009/Notebook/NRP-UEA-Norwich iGEM/2013/07/25
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Streak purified all the isolated single Actinomycete colonies that appeared contaminated.
Many single colony plates were removed due to lack of growth.
All single colony plates were streaked to produce lawns (2 lawns per colony) in preparation for spore stocks.
After the conjugation plates were incubated at 30 °C overnight, they were overlayed with antibiotics - 1 ml of sterile water, 20 ul nalidixic acid and 100 ul kanamycin per plate. The plates were then incubated again overnight at 30 °C.
The two ligation samples from the previous day were analysed by gel electrophoresis fig 2, alongside controls (2b2L and 3b1L) and the samples 2b and 2c from the 22nd July.
Transformation reactions were prepared using SOC media for the ligation reactions, J04450 plasmid and sample 3b from 11/07 (J04450 with Nde1 site).
The membrane was retrieved from the shaker and washed with TBST and TBS solutions respectively for ten minutes. It was then incubated in Anti His HRP conjugate solution. After further washing the membrane was imaged using chemiluminescent detection. This gave no visible result.