User:Daniel Ramirez/Notebook/UNAM Genomics Mexico 2011/2011/09/05: Difference between revisions
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== | ==χρόνος πέρασμα September 5th 2011== | ||
* | ====HydA CAI Optimization Control==== | ||
* Today I make a PCR from the two hydrogenases (HydA), the optimized and the wild-type, that I need to assemble the construction we need in order to test the codon optimization we performed on the coding regions of our genetic cirtuit; in theory, Rhizobium etli CFN42 growth shouldn't be affected by the insertion of the plasmids carrying our system and the expression of these, as the usage of codons of these sequences is similar to those sequences that are naturally highly transcribed (i.e. housekeeping genes). I used a Taq platinum polymerase from Invitrogen for the procedure. I had previously isolated the plasmid that carries the wild-type HydA gene, and given that it was relatively concentrated, I diluted it in a 1:100 manner adding HPLC water to prepare it as the PCR template. Also, I took 1 μl from the stock of HydA-Ferredoxin fusion (just as it came synthesized) and diluted it 1:20 with HPLC water, in order to have it as the optimized HydA template. For these, Paulina had already prepared the primers at the adequate concentration from the stock, they are at 5 μmol/μl. | |||
* The PCR reactions were prepared as follows: | |||
Revision as of 03:27, 25 September 2011
 UNAM Genomics Mexico 2011
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χρόνος πέρασμα September 5th 2011HydA CAI Optimization Control
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