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 .
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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