SESIÓN LABORATORIO 16/07 // LABORATORY SESSION 16/07
16 DE JULIO
Laura, una de las componentes del equipo, se ha ofrecido voluntaria para contaros parte de lo que hemos hecho hoy:
Hoy, para preparar la PCR, hemos mezclado primers liofilizados con una cantidad de agua para conseguir una concentración de 10 picomoles por microlitro. A la vez, hemos doliuido la miniprep del dia anterior 1/100 (DNA molde).
Para realizar esta PCR, hemos usado los primers biobrick F y nitrate R. Tras ello, hemos preparado tubos de PCR, donde hemos mezclado el ADN, el agua, los primers, los nucleótidos, el buffer de la TAQ, y lo hemos llevado todo al termociclador.
Además hemos vuelto a preparar gel de agarosa 2%.
Tras un ratillo de espera, hemos realizado la electroforesis de la mezcla anterior.
Además, Diego también se ha animado a informaros sobre nuestro día:
En esta sesión hemos modificado el plásmido de nuestras células. En primer lugar, y para ponernos en contexto hemos encontrado en el plásmido los genes que nos interesan: (de izquierda a derecha):
Prefijo-Promotor-RBS-Gen GFP-Sufijo
*RBS= Ribosome Binding Site🡪 secuencia de nucleótidos cadena arriba del codón de inicio de un transcrito de ARNm que es responsable del reclutamiento de un ribosoma durante el inicio de la traducción de la proteína.
*Promotor inducible por nitrato.
De esta forma buscamos intercambiar el Gen GFP (da fluorescencia) por otro de color, mediante PCR, para expresar este cuando las células detecten presencia metálica en el medio a analizar.
De la PCR obtendremos la misma cadena (complementaria) sin el GFP en la cual la cola es el sufijo.
Después, utilizando el cultivo realizado el día anterior mediante agotamiento por estrías hemos picado una colonia a LB cloranfenicol (3mL), lo cual se denomina preinóculo, y la hemos dejado overnight a 37 grados.''
In this session we modified the plasmid of our cells. Firstly, as a background,, we found in the plasmid the genes that interest us: (from left to right):
Prefix-Promoter-RBS-Gen GFP-Suffix
* RBS = Ribosome Binding Site nucleotide sequence upstream of the start codon of an mRNA transcript that is responsible for the recruitment of a ribosome during the start of translation of the protein. * Nitrate-inducible promoter.
In this way we seek to exchange the GFP gene (fluorescence) by another color, by PCR, to express it when the cells detect metallic presence in the medium to be analyzed. From the PCR we will obtain the same (complementary) chain without the GFP in which the tail is the suffix.
Using the culture carried out the previous day by striae depletion, we chopped a colony to LB chloramphenicol (3 ml), which is called pre-inoculum, and left it overnight at 37 degrees.
Laura, one of the members of the team, has been voluntary to tell you what we have done:
''Today, to prepare the PCR, we have mixed lyophilized primers with a quantity of water to achieve a concentration of 10 picomoles per microliter. At the same time, we have worked the miniprep from the previous day 1/100 (DNA mold).
To perform this PCR, we have used the primers 'biobrick F' and 'nitrate R'. After that, we have prepared PCR tubes, where we have mixed the DNA, the water, the primers, the nucleotides and the buffer of the TAQ, and we have taken everything to the thermal cycler.
We have also returned to prepare 2% agarose gel.
After a while of waiting, we have performed the electrophoresis of the previous mixture.''
Diego also wanted to tell you about our day:
''In this sesión, we also have modified the plasmid of our cells. Firstly, as a background,, we found in the plasmid the genes that interest us: (from left to right):
Prefix-Promoter-RBS-Gen GFP-Suffix
* RBS = Ribosome Binding Site nucleotide sequence upstream of the start codon of an mRNA transcript that is responsible for the recruitment of a ribosome during the start of translation of the protein. * Nitrate-inducible promoter.
In this way we seek to exchange the GFP gene (fluorescence) by another color, by PCR, to express it when the cells detect metallic presence in the medium to be analyzed. From the PCR we will obtain the same (complementary) chain without the GFP in which the tail is the suffix.
Using the culture carried out the previous day by striae depletion, we chopped a colony to LB chloramphenicol (3 ml), which is called pre-inoculum, and left it overnight at 37 degrees.