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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

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Week Six (19/06/2008)

With an agarose gel ran the 4 colonies that were mutated so as to confirm the mutation of the colonies planted earlier. And given that 3 colonies were mutated and a given mixture of cells. This was done by using 2 primers that allow to know which mutated through the gel. Sabrina
After we explained what we were going to do next week.

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Week Five (12/06/08)

We check each cell mutations by a PCR and running on agarose gel , we obtained positive product with primers used for mutant colonies and some other mixture of cells, so seeded in a petri plates for more colonies and to confirm a mutation. The mutation is done by homologous recombination with a fragment of DNA containing a selectable marker URA 3. Then with specific primers for cells type wild and mutant cells I can see if there is mutation.

Ampicillin sterilize by filtering technique in a laminar flow and placing tubes in 20 to save and not to deteriorate the pregnant thawing and freezing all the time. The ampicillin comes in 1000x concentration (0.1 g / ml).

prepare a growth medium with:
1% yeast extract 2% peptone

2% Glucose Agar
2% (to prepare plates).

The image is of PCR and plasmid last week.
in the first well is the marker and through the well of the PCR first the positive and then negative (as there is nothing we can say that there was no contamination), and the last is that of the plasmid .
the person selling the marker delivery also with a paper showing how it will run and the bands that show sizes as base pairs. So we know how many base pairs have the PCR and plasmid .

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Fourth week (06/05/2008)

plasmids extracted from the bacteria E. Coli of our environment LB, precisely following the proper protocol steps: We

an aliquot of 5 ml of our culture of bacteria E. Coli and centrifuged, leaving us with the pellet (the supernatant is composed of medium components and the pellet are bacteria) added a
Buffer has EDTA (by chelating action helps to further destabilize the bacterial membrane) also has a blue dye, Lyse Blue, (which tells me when everything is homogenized). Add another solution
Buffer (comprising NaOH and SDS ). There is a basic analysis, and color change from clear to blue. Be mixed by immersion, because everything I have inside of the tube is too sensitive, until the blue color is the same everywhere. We added the third
buffer which lowers the pH favoring renaturation of DNA . but, as an abrupt change is renature wrong and what I get is a whole precipitate membrane debris, walls genomic DNA etc., the dye changes from blue to colorless (dip mix again.) As the plasmid
is very small and it is linked physically , not separated and can renature well. I have left in solution.
centrifuged.
We put the solution in a tall column where you will be united in the Recina DNA, passing through it (enzymes, etc). Centrifuge.
We wash with Buffer separating proteins and salts plasmid. Centrifuge. He adds another
Buffer to continue washing and purifying our plasmid. Centrifuged. Buffer place another that will take off the plasmid the tall column (centrifuging to ensure that under all purified plasmid).

prepare an agarose gel 1% w / v in 45 ml
placed in the wells of the gel three PCR and plasmid solution (each with 1.25 ul of loading buffer (which by its density, contains glycerin, helps us do what we sow sow down to posillo and not left floating, and the dye that is allows us to see the front fluently).

Next week we will see the results of another agarose gel .

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Third week (29/05/2008 )

amplified DNA sequence of 800 bp promoter UGA -3. For this we use the PCR technique , components to accomplish this are:
Buffer dNTP

MgCl 2 First

DNA pol ( Taq polymerase , thermostable , survive high temperatures)
mold genomic DNA

H2O concentrations calculated to ensure that all of this in 150 ul

* A mix when everything is important to note that the first thing you should put the water and the B uffer because other components are very sensitive to place in the vacuum tube and nothing happens to them.

prepare three tubes with 50 ul PCR each mixture. Two of them with the mold (we will call positive (+)) and one without it (let's call this negative (-)). Tube is necessary to negative because it indicates whether there was contamination or not and whether what I have in the tubes is only gene DNA gel UGA also -3 or more. We want to ensure only be amplifying the selected sequence.

To make PCR is a program where everything is heated to 94 ° C (to separate DNA chains ) lowering the temperature to 63 ° C (at this temperature are joined primers) and then rises to 72 ° C (the temperature optimum for polymerization ). This program is done in cycles amplify and have a lot of product.

Program used: 94 ° C 5

min 94 ° C 1 min \\
63 ° C 1 min ) is repeated 30 times.
72 ° C 2 min /
72 ° C 5 min