User:Daniel Ramirez/Notebook/UNAM Genomics Mexico 2011/2011/09/02

UNAM Genomics Mexico 2011

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χρόνος πέρασμα September 2nd 2011 HydA CAI Optimization Control ABSTRACT Plasmid extraction of the bioparts J04500 and B0015. Double digestion of these sequences. Today I utilized Roche's Plasmid Extraction Kit to isolate the plasmids pSB1AK3 that carry the bioparts J04500 and B0015. The bacteria were left growing for seven hours at 37°C. As we were running out of spinning tubes, and as because somehow Fabricio López had the hunch that another kit's spinning tubes might leave more concentrated the plasmid isolation, I did the extraction using both spinning tubes to compare their performance. I then ran a 1% agarose electrophoretic gel to see the plasmid extractions, 130 V 35 minutes. Lane #1 contains a 250 bp Ladder, lanes #2 and #4 contain the B0015 and J04500 plasmid extraction using the spinning tubes from the same kit as the roche's plasmid extraction kit respectively, lanes #3 and #5 contain the B0015 and J04500 plasmid extraction using the spinning tubes from another kit. The other kit's spinning tubes are clearly superior in the fact that they elute much more plasmid. As these bioparts are to be ligated with the optimized and the wild-type HydA CDS sequences, they need to be cut. This is how the double digestions were performed: Reactant Volume H20 12 μl Buffer #2 3 μl BSA 10x 2 μl PstI 1.5 μl SpeI 1.5 μl Total 30 μl For the digestion of J04500 Reactant Volume H20 12 μl Buffer #4 3 μl BSA 10x 2 μl EcoRI 1.5 μl XbaI 1.5 μl Total 30 μl For the digestion of B0015 The digestions were left at 37°C overnight.