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 using the plasmid vector worked in the first trial. However, the cloning using pSB1C3 was more difficult than we expected and only worked after four trials and extensive optimization of our protocols. We have not been able to pinpoint the problems.
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