More plasmid extraction of the bioparts E0430, J04450 and I20260. The double digestion on E0430 and J04450 was succesful. B0015 transcriptional terminator characterization.
Today I checked the double digestion performed on the plasmids that carry the bioparts E0430 and J04450. As I realized that I will be needing to extract the digestion fragments from the electrophoretic gel itself, I extracted much more plasmid from E.coli cells that carry the bioparts E0430, J04450 and I20260; as the protocol that I will be using to extract from electrophoretic gels says that a lot of plasmid is lost during the procedure.
Lane #1 contains 1kb DNA ladder, lanes #2 and #3 contain the EcoRI-HF and XbaI double digestion on E0430, lanes #4 and #5 contain the EcoRI-HF and SpeI double digestion on J04450, lanes #6 and #8 contain the plasmid extraction of E0430, lanes #9 and #10 contain the plasmid extraction of I20260 and lanes #11 and #12 contain the plasmid extraction of J04450. Enrique Paz will extract the plasmid that carry the biopart E0422 and will give it to me.
The most lower band of lanes #4 and #5 represent the digested fragment of the biopart J04450. This fragment is to be ligated to the fragment that contains E0430 (lane #2, I will not use lane #3 as it is not as clean). The problem with this assembly is that both of the plasmid that contain the bioparts carry the same antibiotic resistance cassette, ampicillin. To avoid the the fact of seeing red colonies (the expected phenotype of the final construction) as a consequence of just the two fragments digested by EcoRI-HF and SpeI being re-ligated, I shall extract from the electrophoretic gel the band of the desired fragment, I will perform this tomorrow with Enrique Paz.
UNAM Genomics Mexico 2011
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