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week (07/17/2008)

Growth prepare three media: LB

Middle (for growing bacteria):
· Extract 0.5% yeast
·
· NaCl 1% 1% Peptone Agar 2%
·

YPD medium (rich medium to grow yeast)
• And (yeast extract) 1%
· P (peptone) 2%
· D (dextrose / glucose) 2% 2% Agar
·

Middle YNB-DO (URA minimal medium (-) to grow yeast)
· YNB (yeast nitrogen base) 0.67%
· Glucose 2% Drop out
· URA (-) 0.19% Agar 2.5%
·

All agar media were added because we do plates.
Autoclaving the media along with tips and Erlenmeyer flasks to sterilize.
prepare 15 plates with media, 5 plates for each and labeled.
prepare a buffer that was needed in the laboratory, Z buffer used, inter alia, for the testing of B-gal two weeks ago. We weighed
components as we did with media, but without autoclaving. Susana
explained as primers are constructed and gave us a problem to do.

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Ninth week (10/07/2008)

We found what we were wrong last time, and after going back to Excel all we realized that the mistake he had was some brackets in the equations to get Miller units.

With the corrected data, we figure in a special program and found that it was actually correct as is the experience.

Sabrina gave us a method protocol Chip (chromatin immunoprecitation) in English, to familiarize with the vocabulary and be able to deduce and understand what each step was and what was required.
This technique consists basically of:
· Fix or leave everything static (covalently joining if any, binding to the protein) using formaldehyde
§ comparing sonicated to break DNA into smaller fragments
· is added to a protein that helps clean up the proteins that are more
· is puts an antibody that attaches to the protein that interests me
§ comparing add a ball of protein that is also attached to the antibody and this precipitated PCR
· is made and analyzes the content.

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Eighth week (07/03/1908 )

reporter gene assay, using the method of Miller, we found the activity of the b-galactosidase.
substrate was added to samples containing other compounds to see if these interfere with the activity of the enzyme, the product that gives this reaction with ONPG is yellow and is measured in a spectrophotometer. We
aliquot tubes last week and we add Buffer Z. SDS and chloroform to put that then to put ONPG, this substance can come, adding that, the b-galactosidase to catalyze the reaction starts and when the product (the yellow) it stops with Na2CO3 and placed in cold. (We duplicate samples and every 30 seconds to add ONPG tubes). We note
initial time and final time of the reaction. We take the cell density of the surplus ml tubes at an absorbance at 570 nm. Absorbance for Miller units at 420 nm. Make a table that includes all values \u200b\u200band also a chart appears in Miller units versus time.

While we thought we had worked well and the absorbance were quite successful in the time to make the graphics got very poorly and a straight illogical.

Figure if we had done well would have been something like

We

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seventh week (26/06/08)

induction.
Day 1: take a colony of yeast containing a plasmid (YEP -357) and placed on minimal medium, leave it overnight.
Day 2: it is a dilution of the medium and leaving it overnight.
Day 3: the medium is divided into three tubes are centrifuged pellet staying with and adding back the minimal media but cool (one of the three tubes were added Leucine (un aminoácido )). Después de 30 minutos al tubo de los aminoácidos y a otro se le agrega GABA .
Se toma una muestra a tiempo 0 de los tres tubos y otras a los 120 minutos.
El objetivo es saber en que condiciones el gen se induce mejor, si afecta la presencia de aminoácidos en la inducción y como se ven estos cambios según la variación del tiempo.

Sabrina también nos explicó como hacer para saber si una proteína interactúa con el gen UGA 4. para eso se usa formaldehido que une covalentemente a la proteína con el gen. Después un antibody and agarose ball bind with the protein. This is precipitated using the centrifuge.
After that you can have 2 possibilities: that the protein sticks to UGA 4 and rush with this, or stick to something else and is the UGA gene 4 in the supernatant . If UGA is
4 in the supernatant means that the protein does not interact with it.
was verified by PCR