User:Daniel Ramirez/Notebook/UNAM Genomics Mexico 2011/2011/09/21: Difference between revisions
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==χρόνος πέρασμα September 21th 2011== | ==χρόνος πέρασμα September 21th 2011== | ||
====HydA CAI Optimization Control==== | ====HydA CAI Optimization Control==== | ||
=====ABSTRACT===== | |||
* PCR of the HydA optimized CDS using Pfx platinum polymerase from Life Technologies and the PCRx Enhancer 10x Buffer. | |||
== == | |||
* Today I tried another strategy to perform the optimized HydA CDS that I need to proceed with the assembly of the construction that will serve to test if the CAI optimization works properly. I used a Pfx platinum polymerase from Life Technologies as well as a special buffer called PCRx Enhancer 10x Buffer, that is specially used for primers that are long and/or rich in GC. The primers I'm using are both; they are 34 nt long and 61 nt long, with a GC content of 50% and 48%, respectively. The manufacturer's advise is that this buffer should be tried at different concentrations (e.g. 0.5x, 1x, 2x) [http://tools.invitrogen.com/content/sfs/manuals/platinumpfx_pps.pdf]. So I prepared three different sets of reactions that varied with this buffer, and the same set was subjected to a temperature gradient to see what annealing temperature gives the best amplification (and also to test if any secondary structure or primer dimers inferfere with the reaction, greater the temperature, less secondary structures). I also prepared reactions to amplify the wild-type HydA CDS, this served as a positive control, as I have already been able to amplify this region [INSERTAR LINK]. | * Today I tried another strategy to perform the optimized HydA CDS that I need to proceed with the assembly of the construction that will serve to test if the CAI optimization works properly. I used a Pfx platinum polymerase from Life Technologies as well as a special buffer called PCRx Enhancer 10x Buffer, that is specially used for primers that are long and/or rich in GC. The primers I'm using are both; they are 34 nt long and 61 nt long, with a GC content of 50% and 48%, respectively. The manufacturer's advise is that this buffer should be tried at different concentrations (e.g. 0.5x, 1x, 2x) [http://tools.invitrogen.com/content/sfs/manuals/platinumpfx_pps.pdf]. So I prepared three different sets of reactions that varied with this buffer, and the same set was subjected to a temperature gradient to see what annealing temperature gives the best amplification (and also to test if any secondary structure or primer dimers inferfere with the reaction, greater the temperature, less secondary structures). I also prepared reactions to amplify the wild-type HydA CDS, this served as a positive control, as I have already been able to amplify this region [INSERTAR LINK]. | ||
Revision as of 01:18, 25 September 2011
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χρόνος πέρασμα September 21th 2011HydA CAI Optimization ControlABSTRACT
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