Cloning procedures did not work initially due to confounding factors of contamination in media and competent cells, inappropriate design of digestion sites, and non-optimal PCR reactions.
We have attempted to clone our constructs into pSB1C3 and another plasmid vector that has both EcoR1 and Pst1 digestion sites. The cloning into the plasmid vector works at the first trial. However, the cloning into pSB1C3 was more difficult and only worked after four trials and extensive optimization of our protocols.
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