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PRACTICAS DE LABORATORIO DE
BIOLOGIA MOLECULAR
PRACTICA 1
REGLAMENTO DE LA PRACTICA DE LABORATORIO DE BIOLOGIA MOLECULAR
Los estudiantes deben de tener en cuenta lo siguiente para el desarrollo adecuado de las
prcticas de laboratorio:
1.
2.
3.
4.
Exposicin Perctanea: Lave inmediatamente el rea expuesta con agua y jabn germicida;
si la herida est sangrando, apritela o estimule el sangrado, siempre que el rea corporal lo
tolere. Posteriormente, aplique solucin desinfectante despus de concluido el lavado.
Exposicin en Mucosas : Lave profusamente el rea con agua o solucin salina.
Exposicin en Piel No Intacta : Lave el rea profusamente con solucin salina y aplique
solucin antisptica.
Exposicin en Piel Intacta : Lave simplemente el rea con agua y jabn profusamente.
Reportar accidente : Todos los estudiantes y el personal que labore en el laboratorio deben
conocer la importancia de informar inmediatamente una exposicin ocupacional y tener
garantas de la confidencialidad y el respeto con el cual ser tratado.
El reporte se debe hacer dentro de las primeras 24 - 72 horas de presentado el accidente.
Leyenda
Las superficies pueden calentarse durante su
uso.
Esta es capaz de generar corrientes letales.
Utilice las mismas precauciones que con
cualquier dispositivo elctrico. No opere sin la
cubierta en su lugar. No opere en humedad o
ambientes hmedos en los cuales esta pueda
causar dao en los componentes elctricos
internos, no opere con cables conectores con
alambres sueltos
Radiacin ultravioleta que puede ser perjudicial
para la piel y los ojos, no se exponga a esta
radiacin sin proteccin de ocular o de piel
Este
aviso
alerta
potencialmente peligrosas.
de
situaciones
En las etiquetas habr, entre otras cosas, uno o varios smbolos de peligro, as como un
nmero restringido de indicaciones sobre los riesgos y sobre las medidas de prevencin a
adoptar.
La etiqueta le informa inmediatamente sobre el producto y le permite evitar las confusiones
y los errores a la hora de utilizarlo.
La etiqueta es imprescindible en caso de accidente, le dar las indicaciones necesarias
sobre el comportamiento a seguir en caso de accidente.
1 Identificacin del producto (nombre qumico de la sustancia o nombre comercial del
preparado).
2 Composicin (para los preparados relacin de sustancias peligrosas presentes, segn
concentracin y toxicidad).
3 Responsable de la comercializacin (Nombre, direccin y telfono).
4 Identificacin de peligros.
5 Descripcin del riesgo (Frases R*).
6 Medidas preventivas (Frases S*).
Significado
O Comburente
E Explosivo.
Medidas preventivas
Prohibido fumar.
Almacenar
los
productos en un lugar
bien ventilado
Guardar los productos
inflamables
(smbolo
F)
bien
separados
de
los
Productos que pueden favorecer o
productos
comburentes
activar la
(O).
combustin.
No utilizar cerca de una
fuente de calor.
Productos que pueden explotar por Prohibido fumar.
la accin del calor, de un choque o Evitar el exceso de
de un rozamiento
calor y proteger contra
los rayos solares.
No ponerlos cerca de
fuentes
de
calor,
lmparas, radiadores,
etc
Riesgos de alteracin de la salud
Smbolo
Significado
Medidas preventivas
o
problemas
de
R Radioactivo Cancergenos
esterilidad.
Productos peligrosos en caso de
penetracin en el organismo por la nariz,
la boca o a travs de la piel.
T Txico
Productos que pueden alterar el conjunto
del organismo o lesionar nicamente,
ciertos rganos: pulmones, hgado,
corazn, nervios.
T+
C Corrosivo
9. ALMACENAMIENTO
CANCERGENAS.
Prohibido fumar
Utilizar
Equipos
de
Proteccin
Individual
(EPI) para evitar cualquier
contacto.
Trabajar en locales bien
ventilados.
Buena higiene: lavarse
las manos, no comer
durante la utilizacin de
los productos.
Conservar los productos
en el envase de origen.
Tras el uso, lavarse bien
Productos que pueden provocar la la cara y las manos.
destruccin de tejidos vivos..
Verter
siempre
el
Queman la piel y las mucosas, pueden producto en el agua y no
producir lesiones graves que pueden al contrario.
provocar cicatrices.
DE
SUSTANCIAS
TERATOGNAS,
MUTGENICAS
Son aquellas sustancias que estn clasificadas como carcingenos a los humanos o
razonablemente sospechoso como cancerigenos a los humanos; teratgenos o mutagnicas en
las monografas de la Agencia Internacional del Cncer (IARC) o estn catalogadas como tal
por el proveedor de la sustancia.
Almacene las sustancias carcingenas, mutagnicas y teratgenas separadas de otras
sustancias. Identifique en un croquis o un plano de laboratorio la localizacin de estas
sustancias. Limita la cantidad almacenada de estas sustancias a lo estrictamente necesario
para una semana de trabajo. Mantenga un inventario de las sustancias carcingenas en el
laboratorio.
Mantenga al personal femenino en estado de gestacin totalmente aislado de las reas donde
se utilicen estas sustancias.
Los equipos y aparatos nunca deben colocarse en zonas de paso, en particular en los
pasillos del laboratorio.
Todos los aparatos con toma elctrica debern cumplir las normativas de seguridad
correspondientes. Nunca deben utilizarse en zonas mal aisladas y expuestas a la humedad.
Las fuentes de calor (calentadores, termobloques, etc.), sobre todo si se alcanzan
temperaturas elevadas, debern estar debidamente sealizadas para evitar quemaduras
accidentales.
Todos los procedimientos de utilizacin de aparatos deberan contar obligatoriamente con
apartados relativos a su utilizacin segura.
Nevera.
Un adecuado mantenimiento, limpieza y desinfeccin sistemticos de los aparatos reduce
considerablemente los riesgos asociados a su utilizacin. Sin embargo, aun en estas
condiciones, hay que tener en cuenta lo siguiente:
Congelador
La congelacin es un proceso que mantiene la viabilidad de muchos muestras de ADN, de ah
un potencial riesgo y las siguientes recomendaciones:
Incubadoras
La limpieza y la desinfeccin, peridicas y sistemticas, son el mtodo recomendable para
reducir los riesgos derivados de la contaminacin accidental del personal del laboratorio.
Termociclador Y Bao Termorregulado
Los termocicladores constituyen una nueva fuente de accidentes, entre los ms frecuentes
son las explosiones cuando se usan para calentar las muestras, ya que la diferencia de
velocidad de calentamiento produce burbujas que pueden estallar.
Las botellas o matraces deben tener el tapn aflojado, ya que si est cerrado estallan
fcilmente.
Estar siempre presente, con la ropa y pantalla facial adecuadas, y controlar la intensidad del
aparato, que slo puede ser la mxima con agua y la mnima si se usa con gel de agarosa.
Deber existir una tabla bien visible de los tiempos en cada posicin del potencimetro y de
las cantidades a emplear.
Centrfuga
Los mayores riesgos derivan, sobre todo, de la contaminacin por los aerosoles generados
durante la centrifugacin de materiales biolgicos y, en menor medida, de los traumatismos
accidentales. Se recomienda:
PRACTICA 2
ELECTROFORESIS EN GELES DE AGAROSA
La electroforesis en general permite la separacin de molculas como consecuencia de su
diferente movilidad en un campo elctrico. Por lo tanto, el parmetro esencial es la diferente
carga elctrica de las molculas. Es importante recordar que sta depende del pH del medio.
En determinados soportes, como el gel de agarosa mostrado aqu, el medio (gel) ofrece una
resistencia notable al avance de las molculas, por lo cual la movilidad depende tambin del
tamao de la molcula.
Las molculas de DNA y RNA son en general demasiado grandes para que avancen a una
velocidad razonable por estos geles, por lo que lo habitual es fragmentarlas antes de
someterlas a electroforesis.
La agarosa es un polisacrido (originalmente obtenido de algas, como el agar-agar, pero de
composicin ms homognea), cuyas disoluciones (tpicamente de 0,5 a 2%) poseen la
propiedad de permanecer lquidas por encima de aprox. 50C y formar un gel, semislido, al
enfriarse. Este gel est constituido por una matriz o trama tridimensional de fibras polimericas,
embebida en gran cantidad de medio lquido, que retarda el paso de las molculas de cido
nucleico a su travs, en mayor medida cuanto ms grandes sean stas.
Al preparar el gel enfriando la agarosa en un molde adecuado, se dejan en l unos huecos o
pocillos para poder introducir luego en ellos la muestra, y que as sta se vea obligada a
introducirse en el seno del gel cuando apliquemos el campo elctrico (visto de perfil):
separar fragmentos de ADN entre 0,1 y ms de 30 kb para fragmentos ms pequeos que 0,1
kb, los geles de archilamida ofrecen un poder de resolucin netamente mejor.
X174 DNA/Hinf
I
DNA/Hind III
pGEM DNA
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PRACTICA 3
ISOLATION OF GENOMIC DNA FROM BACTERIA
Because of the large size and the fragile nature of chromosomal DNA, it is unlikely that anyone
has ever isolated it in an intact, undamaged form. Several isolation procedures have been
developed that provide DNA in a biologically active form, but this does not mean it is completely
undamaged. These DNA preparations are stable, of high molecular weight and relatively free of
RNA and protein. Here, a general method will be described for the isolation of DNA in a stable,
biologically active form from microorganisms. The procedure outlined is applicable to many
microorganisms and can be modified as necessary.
Designing an isolation procedure for DNA requires extensive knowledge of the chemical stability
of DNA as well as its condition in the cellular environment. Several chemical bonds may be
susceptible to cleavage during the extraction process. The experimental factors that must be
considered and their effects on various structural aspects of intact DNA are outlined below.
1. pH
Hydrogen bonding between the complementary strands is stable between pH 4 and 10.
The phosphodiester linkages in the DNA backbone are stable between pH 3 and 12.
N-glycoside bonds to purine bases (adenine and guanine) are hydrolyzed at pH values of 3
and less.
2. Temperature
There is considerable variation in the temperature stability of the hydrogen bonds in the
double helix, but most DNA will begin to unwind in the range of 80-90'C.
3. Ionic Strength
DNA is most stable and soluble in salt solutions. Salt concentrations of less than 0.1 M
weaken the hydrogen bonding between complementary strands.
4. Cellular Conditions
Before the DNA can be released, the bacterial cell wall must be lysed. The ease with which
the cell wall is disrupted varies from organism to organism. In some cases (yeast), extensive
grinding or sonic treatment is required, whereas in others (B. subtilis), enzyme hydrolysis of the
cell wall is possible.
Several enzymes are present in the cell that may act to degrade DNA, but the most serious
damage is caused by the deoxyribonucleases. These enzymes catalyze the hydrolysis of
phosphodiester linkages.
Native DNA is present in the cell as DNA-protein complexes. The proteins (basic proteins
called histones) must be dissociated during the extraction process.
5. Mechanical Stress on the DNA
Gentle manipulations may not always be possible during the isolation process. Grinding,
shaking, stirring, and other disruptive procedures may cause cleavage (shearing or scission) of
the DNA chains. This usually does not cause damage to the secondary structure of the DNA, but
it does reduce the length of the molecules.
_____________________________________________________________________
10
Now that these factors are understood, a general procedure of DNA extraction may be outlined:
Step 1. Disruption of the cell membrane and release of the DNA into a medium in which it
is soluble and protected from degradation
The isolation procedure described here calls for the use of an enzyme, lysozyme, to disrupt the
cell membrane. Lysozyme catalyzes the hydrolysis of glycosidic bonds in cell wall
carbohydrates, thus causing destruction of the outer membrane and release of DNA and other
cellular components. The medium for solution of DNA is a buffered, saline solution containing
EDTA. DNA, because it is ionic, is more soluble and stable in salt solution than in distilled water.
The EDTA serves at least two purposes. First, it binds divalent metal ions (Cd", Mg 2 +, Mn 2 +)
that could form salts with the anionic phosphate groups of the DNA. Second, it inhibits
deoxyribonucleases that have a requirement for Mg2+ or Mn 2 1. Citrate has occasionally been
used as a chelating agent for DNA extraction; however, it is not an effective agent for binding Mn
2 '. The mildly alkaline medium (pH 8) acts to reduce electrostatic interaction between DNA and
the basic histones and the polycationic amines, spermine and spermidine (see Experiment 21).
The relatively high pH also tends to diminish nuclease activity and denature other proteins.
Step 2. Dissociation of the protein-DNA complexes
Detergents are used at this stage to disrupt the ionic interactions between positively charged
histones and the negatively charged backbone of DNA. Sodium dodecyl sulfate (SDS), an
anionic detergent, binds to proteins and gives them extensive anionic character. A secondary
action of SDS is to act as a denaturant of deoxyribonucleases and other proteins. Also favoring
dissociation of protein-DNA complexes is the alkaline pH, which reduces the positive character
of the histones. To ensure complete dissociation of the DNA-protein complex and to remove
bound cationic amines, a high concentration of a salt (NaCl or sodium perchlorate) is added. The
salt acts by diminishing the ionic interactions between DNA and cations.
Step 3. Separation of the DNA from other soluble cellular components
Before DNA is precipitated, the solution must be deproteinized. This is brought about by
treatment with chloroform/isoamyl alcohol and followed by centrifugation. Upon centrifugation,
three layers are produced:
An upper aqueous phase, a lower organic layer, and a compact band of denatured protein at
the interface between the aqueous and organic phases. Chloroform causes surface
denaturation of proteins. Isoamyl alcohol reduces foaming and stabilizes the interface
between the aqueous phase and the organic phases where the protein collects.
The upper aqueous phase containing nucleic acids is then separated and the DNA
precipitated by addition of ethanol. Because of the ionic nature of DNA, it becomes insoluble
if the aqeuous medium is made less polar by addition of an organic solvent. The DNA forms
a threadlike precipitate that can be collected by "spooling" onto a glass rod. The isolated
DNA may still be contaminated with protein and RNA. Protein can be removed by dissolving
the spooled DNA in saline medium and repeating the chloroform/isoamyl alcohol treatment
until no more denatured protein collects at the interface.
RNA does not normally precipitate like DNA, but it could still be a minor contaminant. RNA may
be degraded during the procedure by treatment with ribonuclease after the first or second
_____________________________________________________________________
11
deproteinization steps. Alternatively, DNA may be precipitated with isopropanol, which leaves
RNA in solution. Removal of RNA sometimes makes it possible to denature more protein using
chloroform/isoamyl alcohol. If DNA in a highly purified state is required, several deproteinization
and alcohol precipitation steps may be carried out. It is estimated that up to 50% of the cellular
DNA is isolated . The average yield is 1 to 2 mg per gram of wet packed cells.
PROTOCOL
1. Inoculate 50 ml liquid culture (e.g. L broth) with E. coli
2. Grow, with shaking, overnight at 37oC (If a water-bath shaker is unavailable, anaerobic
growth (i.e. no shaking) at room temperature will suffice)
3. Centrifuge culture (This should be done relatively gently---i.e. ca.12000 rpm for 5 min. in a
microcentrifuge
4. Resuspend the pellet(s) in 200 l saline-EDTA
5. Optional: to the cell suspension add 10 ml lysozyme and incubate 30 min. at 37oC
6. To the suspension, add:
100 l SDS
100 l NaCl
50 l Proteinase K
Incubate at 60oC for at least 1 hr and up to 24 hr
7. Gently add 2 volumes of freezer-cold ethanol by carefully pouring it down the side of the tube
so that it forms a layer on top of the aqueous suspension. Wait and watch for about 5 min.; the
white, fibrous-looking material that forms at the interface is DNA (along with any other
contaminating large molecules)
8. Carefully "spool" this precipitate onto the glass rod. Place the precipitate into the
microcentrifuge tube; scrape along the side to remove it, if necessary. Care must be taken at
this step not to "lose" the DNA; it can become rather transparent and difficult to see
9. Let the tube sit open in the rack for about 5 min. to let the alcohol evaporate (i.e. when you
no longer can smell the alcohol). Then, carefully add 100l TE buffer1[1] and let the suspension
sit at room temperature overnight or in the refrigerator for several days to allow the DNA to
become completely resuspended.
10. To the suspension, add RNAse and incubate 30 min at 37oC
11. Add an equal volume of chloroform-isoayml alcohol; gently mix the two layers continuously,
by hand, for about 10 min. Centrifuge (about 4000rpm for 2 min in a microcentrifuge;
alternatively, the RNAse reaction and extraction step can be performed in a larger tube suitable
for use in a table top centrifuge)
12. Carefully remove the upper non-aqueous phase, along with the white fluffy material at the
interface (this is primarily denatured protein)
13. The chloroform-isoamyl alcohol extraction step can be performed 2-3 times more, if desired,
until the white interface is no longer
14. Repeat the ethanol precipitation step, outlined above, on the final aqueous phase, including
resuspension of the DNA in TE buffer
Background
The isolation of DNA is one of the more commonly used procedures in many areas of bacterial
physiology, genetics, molecular biology and biochemistry. Purified DNA is required for many
applications such as studying DNA structure and chemistry, examining DNA-protein interactions,
carrying out DNA hyrbridizations, sequencing or PCR, performing various genetic studies or
gene cloning. The isolation of DNA from bacteria is a relatively simple process. The organism
1
_____________________________________________________________________
12
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13
a. Cells must be resuspended in buffer (some protocols call for washing the cells in the buffer--i.e. resuspending and centrifuging---2 or 3 times) in order to have the ionic strength of the
solution compatible with biomolecules, in particular, the salt and pH. The EDTA is a chelating
agent that ties up divalent metal ions; these ions are often cofactors necessary for the action of
DNAses---enzymes present in the cell, which, when released by lysis along with the DNA, can
degrade the DNA.
b. SDS is an ionic detergent which will lyse most cells and denature some proteins.
c. Lysozyme is used to lyse cells resistant to detergent.
d. Raising the ionic strength by the addition of 5M NaCl provides an environment in which DNAprotein interaction, which are often brought about by ionic interactions are thermodynamically
neutralized by the presence of other ions. This helps to dissociate protein from nucleic acid.
e. Proteinase K is a ubiquitous protein-degrading enzyme. It can function in the presence of
detergent and at elevated temperatures. The elevated temperatures are used to denature
DNAses and to facilitate DNA-protein dissociation.
f. Ethanol lowers the effective water concentration, causing large biomolecules to interpenetrate
and aggregate. The result is a visible precipitate at the interface, where the ethanol is
concentrated. As DNA is precipitated and removed, more is exposed to the ethanol and will
precipitate.
g. DNA must become rehydrated completely before it will form a uniform suspension. This can
take a long time, depending on the concentration of the DNA. It can be facilitated by starting off
with low ionic strength buffer and, once resuspended, adding an appropriate amount of
concentrated buffer stock to bring the buffer to the correct ionic strength.
h. Chloroform-isoamyl alcohol is used to deproteinize (it is called the "Sevag procedure" after
the person who first developed and used it in 1938). The chloroform causes surface
denaturation of proteins; the isoamyl alcohol reduces foaming, aids separation and maintains
the stability of the layer of the centrifuged, deproteinized solution.
2. What would you do to show that the precipitating material is really DNA? How would you
quantify the amount?
3. Calculate the maximum amount of DNA expected from this protocol. Assume that the initial
culture had a cell density of 5x109 cells/ml. The E. coli genome is 4.7 Mb long, and the
molecular weight of a nucleotide pair is about 600 daltons.
4. What are the major contaminants in the preparation?
concentrations?
5. If you spill SDS on your skin, nails or hair, would they dissolve? Why?
6. How might you assay for Proteinase K activity?
7. What does lysozyme do in this protocol? Where does it occur naturally and what is its
function?
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14
PRACTICA 4
GENOMIC DNA ISOLATION FROM BLOOD
Genomic DNA isolation is performed according to the FBI protocol. After the blood samples
(stores at -70 C in EDTA vacutainer tubes ) are thawed, standard citrate buffer is added, mixed,
and the tubes are centrifuged. The top portion of the supernatant is discarded and additional
buffer is added, mixed, and again the tube is centrifuged. After the supernatant is discarded, the
pellet is resuspended in a solution of SDS detergent and proteinase K, and the mixture is
incubated at 55 C for one hour. The sample then is phenol extracted once with a
phenol/chloroform/isoamyl alcohol solution, and after centrifugation the aqueous layer is
removed to a fresh microcentrifuge tube. The DNA is ethanol precipitated, resuspended in buffer,
and then ethanol precipitated a second time. After the pellet is dried, buffer is added and the
DNA is resuspended by incubation at 55 C overnight, the genomic DNA solution is assayed by
the polymerase chain reaction.
Protocol
1. Obtain the liquid blood samples in EDTA vacutainer tubes frozen at -70 C.
2. Thaw the frozen samples, add 0.8 ml 1X SSC buffer, and mix. Centrifuge for 1 minute at
12,000 rpm in a microcentrifuge.
3. Remove 1 ml of the supernatant and discard into disinfectant.
4. Add 1 ml of 1X SSC buffer, vortex, centrifuge as above for 1 minute, and remove all of the
supernatant.
5. Add 375 ul of 0.2M NaOAc to each pellet and vortex briefly. Then add 25 l of 10% SDS and 5
ul of proteinase K (20 mg/ml H2O), vortex briefly and incubate for 1 hour at 55 C.
6. Add 120 l phenol/chloroform/isoamyl alcohol and vortex for 30 seconds. Centrifuge the
sample for 2 minutes at 12,000 rpm in a microcentrifuge tube.
7. Carefully remove the aqueous layer to a new 1.5 ml microcentrifuge tube, add 1 ml of cold
100% ethanol, mix, and incubate for 15 minutes at -20 C.
8. Centrifuge for 2 minutes at 12,000 rpm in a microcentrifuge. Decant the supernatant and
drain.
9. Add 180 l 10:1 TE buffer, vortex, and incubate at 55 C for 10 minutes.
10. Add 20 ul 2 M sodium acetate and mix. Add 500 ul of cold 100% ethanol, mix, and centrifuge
for 1 minute at 12,000 rpm in a microcentrifuge.
11. Decant the supernatant and rinse the pellet with 1 ml of 80% ethanol. Centrifuge for 1 minute
at 12,000 rpm in a microcentrifuge.
12. Decant the supernatant, and dry the pellet in a Speedy-Vac for 10 minutes (or until dry).
_____________________________________________________________________
15
13. Resuspend the pellet by adding 200 ul of 10:1 TE buffer. Incubate overnight at 55 C,
vortexing periodically to dissolve the genomic DNA. Store the samples at -20 C.
Solutions
2M NaOAc (sodium acetate):
27.22 g NaOAc-3H2O
ddH2O to 100 ml
20X SSC (standard saline-citrate):
17.53 g
NaCl
8.82 g
sodium citrate
Dissolve in approx. 80 ml ddH2O, adjust pH to 7.0 with hydrochloric acid (HCl) and bring final
volume to 100 ml.
1X SSC (standard saline-citrate):
5 ml
95 ml
100 ml
20X SSC
ddH2O
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16
PRACTICA 5
TRANSFORMACION DE Escherichia coli
Preparacin de clulas competentes
El objetivo de esta prctica es el de preparar las clulas a incorporar el ADN de manera eficaz
(normalmente pocas clulas pueden aceptar ADN exgeno). Primero que todo las clulas son
puestas en cultivo a 37C hasta que la densidad ptica a 600 nm alcance 0.5 (fase de
crecimiento exponencial). Despus son tratadas en un tampn que contiene una fuerte
concentracin de Ca 2+ Despus de este tratamiento las clulas son llamadas competentes para
la transformacin. Las molculas de ADN siendo cargadas negativamente por sus grupos
fosfatos, no pueden aproximarse a las membranas celulares, igualmente cargadas
negativamente (e.g. los grupos fosfatos de los fosfolipidos).
El primer objetivo del Ca2+ es el de neutralizar la carga de la membrana y permitir el contacto
entre el ADN y la clula. Parece igualmente que especies de poros se forman a travs de la
membrana bacteriana, pero este fenmeno permanece poco claro. Como el tamao de un
plsmido que es ha menudo superior al de la clula (un plsmido de 3kb mide alrededor de
m, mientras que la bacteria de E. coli mide entre 1 y 2 m), el proceso de incorporacin es un
fenmeno activo. Despus de la fase de incorporacin, una fase de establecimiento debe tener
lugar. Esta fase es ayudad en su eficacia por un tratamiento de tipo de choque trmico antes de
la incubacin de las bacterias.
MODO OPERATIVO
Todas estas manipulaciones deben hacerse en total esterilidad.
1. Inocular 20 ml de medio de crecimiento LB (que contiene 10 g/l NaCl, 10 g/l triptona y 5 g/l
de extracto de levadura) con 0.5 ml de un precultivo fresco de clulas de E. coli JM109.
2. Incubar a 37C con agitacin (250 ciclos/min) alrededor de 2 horas hasta alcanzar una
densidad ptica de 0.5 a 600 nm.
3. Centrifugar 5 min. a 4000 g
4. Retomar delicadamente los sedimentos bacterianos en 500 l de 0.1 M de CaCl 2, despus
agregar 9 ml de la misma solucin enfriada en el hielo. Dejar 30 min. a 0C.
5. Centrifugar como en (3) y retomar 800 l finales de 0.1 M de CaCl 2. Despus de 1 hora a 0
C las clulas estn listas para la transformacin.
TRANSFORMACION
1. Agregar 5 l de los plasmidos a 200 l de clulas competentes y dejarlas a 45 minutos a 0
C. Preparar un bao a 42 C. Preincubar las cajas de Petri a 37C.
2. Despus de 45 minutos, calentar las clulas 90 segundos a 42C (choque trmico), y
agregar 700 l de medio LB. Dejar 1 hora a 37C . Esta etapa es importante para permitir a
las clulas sintetizar la enzima responsable de la resistencia a la kanamicina
3. Sobre una caja de petri LB-agar conteniendo 100 g/ml de kanamicina, esparcir 40 l de 20
mg/ml de X-Gal con la ayuda de una varilla de vidrio (usar guantes!).
4. Despus de la incubacin a 37C, agregar 10l de IPTG a las clulas transformadas,
mezclar bien y esparcir sobre la caja de petri preparada en el punto 3. Dejar secar alrededor
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17
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18
Ligations Using the pGEM-T and pGEM-T Easy Vectors and the 2X Rapid Ligation
Buffer
Protocol
1. Briefly centrifuge the pGEM-T or pGEM-T Easy Vector and Control Insert DNA tubes to
collect contents at the bottom of the tube.
2. Set up ligation reactions as described below. Note: Use 0.5ml tubes known to have low DNAbinding capacity (e.g., VWR Cat.# 20170-310).
3. Vortex the 2X Rapid Ligation Buffer vigorously before each use.
4. Mix the reactions by pipetting. Incubate the reactions 1 hour at room temperature.
Alternatively, if the maximum number of transformants is required, incubate the reactions
overnight at 4C.
Transformations Using the pGEM-T and pGEM-T Easy Vector Ligation Reactions
Use high efficiency competent cells (1 x 108 cfu/g DNA) for transformations. The ligation of
fragments with a single-base overhang can be inefficient, so it is essential to use cells with a
transformation efficiency of 1 x 10 8 cfu/g DNA (or higher) in order to obtain a reasonable
number of colonies.
We recommend using JM109 High Efficiency Competent Cells (Cat.# L2001); these are
provided with the pGEM-T and pGEM-T Easy Vector Systems II. Other host strains may be
used, but they should be compatible with blue/white color screening and standard ampicillin
selection. JM109 cells should be maintained on M9 minimal medium plates supplemented with
thiamine hydrochloride prior to the preparation of competent cells. This selects for the presence
of the F episome, which carries both the proAB genes, which complement proline auxotrophy in
a host with a ( proAB) deletion, and lacIqZDM15, which is required in the blue/white color
screening process. If you are using competent cells other than JM109 High Efficiency
Competent Cells purchased from Promega, it is important that the appropriate transformation
protocol be followed. Selection for transformants should be on LB/ampicillin/ IPTG/X-Gal plates
(see Section X.A). For best results, do not use plates more than 30 days old.
The genotype of JM109 is recA1, endA1, gyrA96, thi, hsdR17 (rK-,mK+), relA1, supE44, D( lacproAB), [F, traD36, proAB, lacIqZDM15].
____________________________________________________________________________________
19
Protocol
1. Prepare 2 LB/ampicillin/IPTG/X-Gal plates for each ligation reaction, plus two plates for
determining transformation efficiency. Equilibrate the plates to room temperature prior to plating
(Step 10).
2. Centrifuge the tubes containing the ligation reactions to collect contents at the bottom of the
tube. Add 2l of each ligation reaction to (a) sterile 1.5ml microcentrifuge tube(s) on ice (see
Note 1). Set up another tube on ice with 0.1ng uncut plasmid for determination of the
transformation efficiency of the competent cells (see Section III.E).
3. Remove tube(s) of frozen JM109 High Efficiency Competent Cells from 70C storage and
place in an ice bath until just thawed (about 5 minutes). Mix the cells by gently flicking the tube.
4. Carefully transfer 50l of cells into each tube prepared in Step 2 (100l cells for determination
of transformation efficiency).
5. Gently flick the tubes to mix and place them on ice for 20 minutes.
6. Heat-shock the cells for 45-50 seconds in a water bath at exactly 42C (DO NOT SHAKE).
7. Immediately return the tubes to ice for 2 minutes.
8. Add 950l room temperature SOC medium to the tubes containing cells transformed with
ligation reactions and 900l to the tube containing cells transformed with uncut plasmid (LB
broth may be substituted, but colony number may be lower).
9. Incubate for 1.5 hours at 37C with shaking (~150rpm).
10. Plate 100l of each transformation culture onto duplicate antibiotic plates. For the
transformation control, a 1:10 dilution with SOC medium is recommended for plating. If a higher
number of colonies is desired, the cells may be pelleted by centrifugation at 1,000 x gfor 10
minutes, resuspended in 200l of SOC medium, and 100l plated on each of 2 plates.
11. Incubate the plates overnight (1624 hours) at 37C. In our experience, approximately 100
colonies per plate are routinely seen when using competent cells that are 1 x 10 8 cfu/g DNA, if
100l is plated. Longer incubations or storage of plates at 4C (after 37C overnight incubation)
may be used to facilitate blue/white screening. White colonies generally contain inserts;
however, inserts may also be present in blue colonies.
20
PRACTICA 6
AISLAMIENTO DE ADN PLASMIDICO
En el protocolo empleado aqu, las clulas son centrifugadas y resuspendidas en una solucin
iso-osmotica. Las membranas celulares son luego solubilizadas con la ayuda de un detergente
inico (SDS) y el ADN genmico de gran tamao es desnaturalizado por el NaOH, fragmentado
por la accin mecnica del vortex y finalmente concentrado por centrifugacin con la masa
viscosa de desechos bacterianos. El ADN plasmdico es recuperado en el sobrenadante y
purificado por una extraccin fenolica que elimina eficazmente las protenas por
desnaturalizacion (e.g., nucleasas). El ADN recuperado en la fase acuosa es finalmente
precipitado con etanol. Este mtodo se llama lisis alcalina.
MODO OPERATIVO
1. Inocular 5 ml de medio LB +50 g/l ampicilina con una colonia aislada. Incubar durante la
noche a 37C con agitacin.
Transferir 1.5 ml del cultivo en un tubo para microcentrifuga. Centrifugar 1 minuto.
Aspiarar el sobrenadante con una pipeta Pasteur afilada sin aspirar el sedimento de bacterias.
2. Resuspender el sedimento en 100 l de solucin I con la ayuda de un vortex o de la
micropipeta.
SOLUCIN I
50 mM glucose
10 mM EDTA
25 mM Tris-HCl (pH 8.0)
Incubar 5 minutos a temperatura ambiente.
3. Agregar 200 l de solucin II fresca. Cerrar el tubo y mezclar por inversin rpida cinco
veces. No usar el vortex. Incubar en el hielo durante 5 minutos.
SOLUCIN II
0.2 N NaOH
1% SDS
PREPARACION FRESCA
4. Agregar 150 l de solucin III. Cerrar el tubo y agitar al revs con el vortex durante 10
segundos. Incubar 5 minutos en el hielo.
21
SOLUCIN III
3 M Potasio
5 M Acetato
Centrifugar el tubo durante 5 minutos a temperatura ambiente.
5. Transferir el sobrenadante en un tubo par centrifuga. Agregar 500 l de una mezcla de
fenol:cloroformo 1:1 (usar guantes!). vortex 30 segundos y centrifugar durante 2 minutos.
Transferir el sobrenadante delicadamente a un tubo nuevo sin perturbar la interfase de
protenas. (OPCIONAL)
6. Agregar dos volmenes de etanol 100 % frio. Mezclar por inversin y dejar precipitar el ADN
en el hielo durante 5 minutos.
Centrifugar 5 minutos a temperatura ambiente.
7. Botar el sobrenadante y agregar 1 ml de etanol 70% fri. Vortex 5 segundos y recentrifugar.
Botar el sobrenadante y aspirar el resto con la bomba de vaci (pipeta afilada),. Dejar evaporar
las ltimas de trazas de etanol.
22
PRACTICA 7
ANALISIS DE SECUENCIAS
Existen diversas bases datos de secuencias de ADN y protenas, de acceso libre, donde el
investigador puede encontrar la secuencia de interes.
Entre las ms importantes tenemos:
Nucleotide Databases
dbEST
dbGSS
dbSNP
dbSTS
Nucleotide
GenBank
HomoloGene
MGC
PopSet
RefSeq
TPA
Trace Archive
UniGene
UniSTS
Protein Databases
Domains
Proteins
PROW
RefSeq
Structure Databases
Domains
3D Domains
Structure (MMDB)
Taxonomy Databases
Taxonomy
Genome Databases
Genomes
Gene
Expression Databases
GEO
GEO Datasets
LocusLink
COGs
SAGE
Podemos acceder a estas bases de datos entrando a la pagina web de National Center for
Biotechnology Information: http://www.ncbi.nih.gov/, y tecleamos el link de Molecular
Databases.
Dependiendo de la secuencia que estemos buscando, seleccionamos el siguiente link.
EJERCICIO
Vamos a bajar la secuencia del genoma completo del virus Coliphage phiX174, de amplio uso
en biologa molecular como marcador molecular.
Vamos a seleccionar en la siguiente casilla la palabra Genome y escribimos el nombre de la
especie la cual queremos conocer en este caso Coliphage phiX174
PubMed
Search
Entrez
BLAST
for
OMIM
Coliphage phiX174
Books
TaxBrowser
23
Structure
Go
24
COMMENT
REVIEWED REFSEQ: This record has been curated by NCBI staff. The reference
sequence was derived from J02482. [8] intermittent sequences. [15] review; discussion of complete
genome. Double checked with sumex tape.
Single-stranded circular DNA which codes for eleven proteins. Replicative form is duplex, icosahedron,
related to s13 & g4. [21] indicates that mitomycin C reduced with sodium borohydride induced
heat-labile sites in DNA most preferentially at dinucleotide sequence 'gt' (especially 'Pu-g-t').
Bacteriophage phi-X174 single stranded DNA molecules were irradiated with near UV light in the
presence of promazine derivatives, after priming with restriction fragments or synthetic primers [22]. The
resulting DNA fragments were used as templates for in vitro complementary chain synthesis by E.coli
DNA polymerase I [22]. More than 90% of the observed chain terminations were mapped one nucleotide
before a guanine residue [22]. Photoreaction occurred more predominantly with guanine residues
localized in single-stranded parts of the genome [22]. These same guanine residues could also be
damaged when the reaction was performed in the dark, in the presence of promazine cation radicals [22].
FEATURES
source
Location/Qualifiers
1..5386
/organism="Coliphage phiX174"
/mol_type="genomic DNA"
/specific_host="Escherichia coli"
/db_xref="taxon:10847"
gene
join(3981..5386,1..136)
/locus_tag="phiX174_03"
CDS
join(3981..5386,1..136)
/locus_tag="phiX174_03"
/codon_start=1
/transl_table=11
/product="rf replication, viral strand synthesis protein"
/protein_id="NP_040703.1"
/db_xref="GI:9626373"
/translation=
"MVRSYYPSECHADYFDFERIEALKPAIEACGISTLSQSPMLGFHKQMDNRIKLLEEILSFRMQGVE
FDNGDMYVDGHKAASDVRDEFVSVTEKLMDELAQCYNVLPQLDINNTIDHRPEGDEKWFLENE
KTVTQFCRKLAAERPLKDIRDEYNYPKKKGIKDECSRLLEASTMKSRRGFAIQRLMNAMRQAHA
DGWFIVFDTLTLADDRLEAFYDNPNALRDYFRDIGRMVLAAEGRKANDSHADCYQYFCVPEYG
TANGRLHFHAVHFMRTLPTGSVDPNFGRRVRNRRQLNSLQNTWPYGYSMPIAVRYTQDAFSRSG
WLWPVDAKGEPLKATSYMAVGFYVAKYVNKKSDMDLAAKGLGAKEWNNSLKTKLSLLPKKLF
RIRMSRNFGMKMLTMTNLSTECLIQLTKLGYDATPFNQILKQNAKREMRLRLGKVTVADVLAAQ
PVTTNLLKFMRASIKMIGVSNLQSFIASMTQKLTLSDISDESKNYLDKAGITTACLRIKSKWTAGG
K"
gene
join(4497..5386,1..136)
/locus_tag="phiX174_04"
CDS
join(4497..5386,1..136)
/locus_tag="phiX174_04"
/codon_start=1
/transl_table=11
/product="shut off host DNA synthesis protein"
/protein_id="NP_040704.1"
/db_xref="GI:9626374"
/translation=
"MKSRRGFAIQRLMNAMRQAHADGWFIVFDTLTLADDRLEAFYDNPNALRDYFRDIGRMVLAAE
GRKANDSHADCYQYFCVPEYGTANGRLHFHAVHFMRTLPTGSVDPNFGRRVRNRRQLNSLQNT
25
WPYGYSMPIAVRYTQDAFSRSGWLWPVDAKGEPLATSYMAVGFYVAKYVNKKSDMDLAAKGL
GAKEWNNSLKTKLSLLPKKLFRIRMSRNFGMKMLTMTNLSTECLIQLTKLGYDATPFNQILKQNA
KREMRLRLGKVTVADVLAAQPVTTNLLKFMRASIKMIGVSNLQSFIASMTQKLTLSDISDESKNY
LDKAGITTACLRIKSKWTAGGK"
gene
join(5075..5386,1..51)
/locus_tag="phiX174_05"
CDS
join(5075..5386,1..51)
/locus_tag="phiX174_05"
/codon_start=1
/transl_table=11
/product="capsid morphogenesis protein"
/protein_id="NP_040705.1"
/db_xref="GI:9626375"
/translation=
"MEQLTKNQAVATSQEAVQNQNEPQLRDENAHNDKSVHGVLNPTYQAGLRRDAVQPDIEAERKK
RDEIEAGKSYCSRRFGGATCDDKSAQIYARFDKNDWRIQPAEFYRFHDAEVNTFGYF"
variation
23
/locus_tag="phiX174_05"
/note="c in wt; t in am18 and am35 [14]"
variation
25
/locus_tag="phiX174_05"
/note="g in wt; c in ts116 [14]"
gene
51..221
/locus_tag="phiX174_06"
CDS
51..221
/locus_tag="phiX174_06"
/codon_start=1
/transl_table=11
/product="gene K protein"
/protein_id="NP_040706.1"
/db_xref="GI:9626376"
/translation=
"MSRKIILIKQELLLLVYELNRSGLLAENEKIRPILAQLEKLLLCDLSPSTNDSVKN"
variation
57
/locus_tag="phiX174_06"
/note="c in wt; t in am6 [14]"
variation
117
/locus_tag="phiX174_06"
/note="g in wt; a in am6 [14]"
gene
133..393
/locus_tag="phiX174_07"
CDS
133..393
/locus_tag="phiX174_07"
/codon_start=1
/transl_table=11
/product="DNA maturation protein"
/protein_id="NP_040707.1"
/db_xref="GI:9626377"
/translation=
26
"MRKFDLSLRSSRSSYFATFRHQLTILSKTDALDEEKWLNMLGTFVKDWFRYESHFVHGRDSLVDI
LKERGLLSESDAVQPLIGKKS"
gene
358..3975
/locus_tag="phiX174_02"
mRNA
358..3975
/locus_tag="phiX174_02"
/note="mRNA (major alt.)"
gene
358..991
/locus_tag="phiX174_01"
mRNA
358..991
/locus_tag="phiX174_01"
/note="mRNA (minor alt.)"
CDS
390..848
/locus_tag="phiX174_01"
/codon_start=1
/transl_table=11
/product="capsid morphogenesis protein"
/protein_id="NP_040708.1"
/db_xref="GI:9626378"
/translation=
"MSQVTEQSVRFQTALASIKLIQASAVLDLTEDDFDFLTSNKVWIATDRSRARRCVEACVYGTLDF
VGYPRFPAPVEFIAAVIAYYVHPVNIQTACLIMEGAEFTENIINGVERPVKAAELFAFTLRVRAGNT
DVLTDAEENVRQKLRAEGVM"
gene
568..843
/locus_tag="phiX174_08"
CDS
568..843
/locus_tag="phiX174_08"
/codon_start=1
/transl_table=11
/product="cell lysis protein"
/protein_id="NP_040709.1"
/db_xref="GI:9626379"
/translation=
"MVRWTLWDTLAFLLLLSLLLPSLLIMFIPSTFKRPVSSWKALNLRKTLLMASSVRLKPLNCSRLP
CVYAQETLTFLLTQKKTCVKNYVRKE"
gene
848..964
/locus_tag="phiX174_09"
CDS
848..964
/locus_tag="phiX174_09"
/codon_start=1
/transl_table=11
/product="core protein, DNA condensation protein"
/protein_id="NP_040710.1"
/db_xref="GI:9626380"
/translation=
"MSKGKKRSGARPGRPQPLRGTKGKRKGARLWYVGGQQF"
CDS
1001..2284
/locus_tag="phiX174_02"
27
/codon_start=1
/transl_table=11
/product="major coat protein"
/protein_id="NP_040711.1"
/db_xref="GI:9626381"
/translation=
"MSNIQTGAERMPHDLSHLGFLAGQIGRLITISTTPVIAGDSFEMDAVGALRLSPLRRGLAIDSTVDI
FTFYVPHRHVYGEQWIKFMKDGVNATPLPTVNTTGYIDHAAFLGTINPDTNKIPKHLFQGYLNIY
NNYFKAPWMPDRTEANPNELNQDDARYGFRCCHLKNIWTAPLPPETELSRQMTTSTTSIDIMGLQ
AAYANLHTDQERDYFMQRYHDVISSFGGKTSYDADNRPLLVMRSNLWASGYDVDGTDQTSLGQ
FSGRVQQTYKHSVPRFFVPEHGTMFTLALVRFPPTATKEIQYLNAKGALTYTDIAGDPVLYGNLPP
REISMKDVFRSGDSSKKFKIAEGQWYRYAPSYVSPAYHLLEGFPFIQEPPSGDLQERVLIRHHDYD
QCFQSVQLLQWNSQVKFNVTVYRNLPTTRDSIMTS"
gene
2395..2922
/locus_tag="phiX174_10"
CDS
2395..2922
/locus_tag="phiX174_10"
/codon_start=1
/transl_table=11
/product="major spike protein"
/protein_id="NP_040712.1"
/db_xref="GI:9626382"
/translation=
"MFQTFISRHNSNFFSDKLVLTSVTPASSAPVLQTPKATSSTLYFDSLTVNAGNGGFLHCIQMDTSV
NAANQVVSVGADIAFDADPKFFACLVRFESSSVPTTLPTAYDVYPLNGRHDGGYYTVKDCVTIDV
LPRTPGNNVYVGFMVWSNFTATKCRGLVSLNQVIKEIICLQPLK"
gene
2931..3917
/locus_tag="phiX174_11"
CDS
2931..3917
/locus_tag="phiX174_11"
/codon_start=1
/transl_table=11
/product="minor spike protein, adsorption"
/protein_id="NP_040713.1"
/db_xref="GI:9626383"
/translation=
"MFGAIAGGIASALAGGAMSKLFGGGQKAASGGIQGDVLATDNNTVGMGDAGIKSAIQGSNVPN
PDEAAPSFVSGAMAKAGKGLLEGTLQAGTSAVSDKLLDLVGLGGKSAADKGKDTRDYLAAAFP
ELNAWERAGADASSAGMVDAGFENQKELTKMQLDNQKEIAEMQNETQKEIAGIQSATSRQNTK
DQVYAQNEMLAYQQKESTARVASIMENTNLSKQQQVSEIMRQMLTQAQTAGQYFTNDQIKEMTR
KVSAEVDLVHQQTQNQRYGSSHIGATAKDISNVVTDAASGVVDIFHGIDKAVADTWNNFWKDGK
ADGIGSNLSRK"
misc_feature 3962
/locus_tag="phiX174_02"
/note="transcription start site"
rep_origin
4306
/locus_tag="phiX174_03"
/note="origin of viral strand synthesis"
misc_feature 4899
/locus_tag="phiX174_04"
/note="transcription start site"
ORIGIN
1 gagttttatc gcttccatga cgcagaagtt aacactttcg gatatttctg atgagtcgaa
61 aaattatctt gataaagcag gaattactac tgcttgttta cgaattaaat cgaagtggac
121 tgctggcgga aaatgagaaa attcgaccta tccttgcgca gctcgagaag ctcttacttt
181 gcgacctttc gccatcaact aacgattctg tcaaaaactg acgcgttgga tgaggagaag
241 tggcttaata tgcttggcac gttcgtcaag gactggttta gatatgagtc acattttgtt
301 catggtagag attctcttgt tgacatttta aaagagcgtg gattactatc tgagtccgat
361 gctgttcaac cactaatagg taagaaatca tgagtcaagt tactgaacaa tccgtacgtt
421 tccagaccgc tttggcctct attaagctca ttcaggcttc tgccgttttg gatttaaccg
481 aagatgattt cgattttctg acgagtaaca aagtttggat tgctactgac cgctctcgtg
541 ctcgtcgctg cgttgaggct tgcgtttatg gtacgctgga ctttgtggga taccctcgct
601 ttcctgctcc tgttgagttt attgctgccg tcattgctta ttatgttcat cccgtcaaca
661 ttcaaacggc ctgtctcatc atggaaggcg ctgaatttac ggaaaacatt attaatggcg
721 tcgagcgtcc ggttaaagcc gctgaattgt tcgcgtttac cttgcgtgta cgcgcaggaa
781 acactgacgt tcttactgac gcagaagaaa acgtgcgtca aaaattacgt gcggaaggag
841 tgatgtaatg tctaaaggta aaaaacgttc tggcgctcgc cctggtcgtc cgcagccgtt
901 gcgaggtact aaaggcaagc gtaaaggcgc tcgtctttgg tatgtaggtg gtcaacaatt
961 ttaattgcag gggcttcggc cccttacttg aggataaatt atgtctaata ttcaaactgg
1021 cgccgagcgt atgccgcatg acctttccca tcttggcttc cttgctggtc agattggtcg
1081 tcttattacc atttcaacta ctccggttat cgctggcgac tccttcgaga tggacgccgt
1141 tggcgctctc cgtctttctc cattgcgtcg tggccttgct attgactcta ctgtagacat
1201 ttttactttt tatgtccctc atcgtcacgt ttatggtgaa cagtggatta agttcatgaa
1261 ggatggtgtt aatgccactc ctctcccgac tgttaacact actggttata ttgaccatgc
1321 cgcttttctt ggcacgatta accctgatac caataaaatc cctaagcatt tgtttcaggg
1381 ttatttgaat atctataaca actattttaa agcgccgtgg atgcctgacc gtaccgaggc
1441 taaccctaat gagcttaatc aagatgatgc tcgttatggt ttccgttgct gccatctcaa
1501 aaacatttgg actgctccgc ttcctcctga gactgagctt tctcgccaaa tgacgacttc
1561 taccacatct attgacatta tgggtctgca agctgcttat gctaatttgc atactgacca
1621 agaacgtgat tacttcatgc agcgttacca tgatgttatt tcttcatttg gaggtaaaac
1681 ctcttatgac gctgacaacc gtcctttact tgtcatgcgc tctaatctct gggcatctgg
1741 ctatgatgtt gatggaactg accaaacgtc gttaggccag ttttctggtc gtgttcaaca
1801 gacctataaa cattctgtgc cgcgtttctt tgttcctgag catggcacta tgtttactct
1861 tgcgcttgtt cgttttccgc ctactgcgac taaagagatt cagtacctta acgctaaagg
1921 tgctttgact tataccgata ttgctggcga ccctgttttg tatggcaact tgccgccgcg
1981 tgaaatttct atgaaggatg ttttccgttc tggtgattcg tctaagaagt ttaagattgc
2041 tgagggtcag tggtatcgtt atgcgccttc gtatgtttct cctgcttatc accttcttga
2101 aggcttccca ttcattcagg aaccgccttc tggtgatttg caagaacgcg tacttattcg
2161 ccaccatgat tatgaccagt gtttccagtc cgttcagttg ttgcagtgga atagtcaggt
2221 taaatttaat gtgaccgttt atcgcaatct gccgaccact cgcgattcaa tcatgacttc
2281 gtgataaaag attgagtgtg aggttataac gccgaagcgg taaaaatttt aatttttgcc
2341 gctgaggggt tgaccaagcg aagcgcggta ggttttctgc ttaggagttt aatcatgttt
2401 cagactttta tttctcgcca taattcaaac tttttttctg ataagctggt tctcacttct
2461 gttactccag cttcttcggc acctgtttta cagacaccta aagctacatc gtcaacgtta
2521 tattttgata gtttgacggt taatgctggt aatggtggtt ttcttcattg cattcagatg
2581 gatacatctg tcaacgccgc taatcaggtt gtttctgttg gtgctgatat tgcttttgat
28
29
30
PRACTICA 8
ENZIMAS DE RESTRICCIN
Las enzimas de restriccin, tambin conocidas como endonucleasas, son enzimas que cortan
los enlaces fosfodiester del material gentico a partir de una secuencia que reconocen.
Las mismas permiten cortar DNA de hebra doble, donde reconocen secuencias palindrmicas
(secuencias que se leen igual en ambas direcciones).
Son extradas de organismos procariticos (bacterias), donde actan como un mecanismo de
defensa, para degradar material gentico extrao que entre en la clula. Las bacterias tienen la
capacidad de metilar su DNA, lo cual sirve para distinguir entre el DNA extrao y el DNA propio.
Las enzimas de restriccin no pueden cortar DNA metilado, de este modo solo afectan el DNA
extranjero y no el DNA bacterial.
Existen 3 tipos de enzimas de restriccin:
Tipo I y Tipo III:
a. Tienen actividad de restriccin (cortan) y modificacin (metilan).
b. Cortan a cierta distancia de la secuencia de reconocimiento, las Tipo I cortan lejos de la
secuencia de reconocimiento, ya sea ro arriba o ro abajo. Las Tipo III cortan de 5-8 bases
antes o despes de la secuencia que reconocen.
c. Necesitan ATP para moverse a travs de la molcula de DNA, desde el lugar de
reconocimiento hasta el sitio del corte.
Tipo II:
a. Slo tienen actividad de restriccin.
b. Cortan de manera consistente y predecible dentro de la secuencia que reconocen.
c. Slo requieren Mg++ como cofactor.
d. No necesitan ATP.
31
Las endonucleasas se nombran a partir de las bacterias de las que son extradas, su nombre
est dado segn el gnero y la especie de la bacteria de donde se aisl por primera vez esta
enzima. La primera letra representa el gnero de la bacteria, las prximas dos indican la
especie, una cuarta letra indica la cepa, y un nmero al final indica la cantidad de enzimas que
se han aislado de esa cepa.
Ejemplo:
Eco RI E = gnero Escherichia
co = especie coli
R = cepa RV 13
I = primera endonucleasa aislada de esta cepa
Las enzimas de restriccin al cortar el DNA pueden producir 2 tipos de cortes:
1. Cohesivos o pegajosos:
2.
GAATTC
CTTAAG
GGATCC
CCTAGG
EcoRI
BamHI
AAGCTT
AACGAA
HindIII
Abruptos o romos:
AATATT
TTATAA
CCCGGG
GGGCCC
SspI
SmaI
Existen varios factores que son crticos al trabajar con enzimas de restriccin y que pueden
afectar la actividad de las mismas:
Pureza del DNA la reaccin de enzimas es muy dependiente de la pureza, contaminantes
como protenas, fenol, cloroformo, etanol, EDTA, SDS, altas concentraciones de sal, etc.
inhiben la endonucleasa.
Temperatura y pH las enzimas son muy sensitivas a temperatura y pH en lo que respecta
a su estabilidad y actividad.
DNAsas Las DNAsas degradan el DNA en presencia de Mg++
Contaminantes con carga (-).
DNA contaminado con otro DNA.
Grados de metilacin algunas endonucleasas son inhibidas por metilacin.
Tipo de molcula de DNA si el DNA no tiene la secuencia que es reconocida por la enzima
accesible, esta no puede cortar el material gentico (ej. si el DNA est superenrrollado el lugar
de restriccin no va a estar accesible para la enzima).
Buffer adecuado este provee el ambiente que necesita la enzima para trabajar en
condiciones ptimas.
32
Importante: Una unidad de enzima se define como la cantidad de enzima que se necesita para
cortar 1g de DNA en 1 hora.
El buffer siempre debe aadirse como un 10% de la reaccin total de digestin.
Secuencias de reconocimiento de algunas enzimas de restriccin
Enzima de restriccin
Organismo de origen
Secuencia de reconocimiento
Eco RI
Escherichia coli RY 13
5'...GA A T T C...3'
Bam HI
Bacillus amyloliquefaciens
5'...GG A T C C...3'
Him III
Haemophillus influenzae Rd
5'...AA G C T T...3'
Hpa I
Haemophillus parainfluenzae
5'...G T TA A C...3'
Not I
Nocardia otitidis-caviarum
5'...G CG G C C G C...3'
Pst I
Providencia stuartii
5'...C T G C AG...3'
Sma I
Serratia marcescens
5'...C C CG G G...3'
BsuRI
Bacillus subtilis
5'...G GC C...3'
Protocolo
I. Digestin de DNA con Enzimas de Restriccin:
1l de buffer 10 X
1lde enzima (EcoRI) (1 unidad por 1 g de DNA)
1L de DNA (1g)
7L de H2O
10L de Volumen Total
Incubar a 37C por 1 hora
II. Preparacin de muestras para la corrida:
DNA sin cortar
1L de DNA sin cortar
9L de H2O
2L de loading dye
DNA cortado
10L de digestin
2L de loading dye
III. Corrida del Gel de Electroforesis:
RESPONDA
1. Mencione 3 factores que afectan la actividad de las enzimas de restriccin.
2. Las enzimas de restriccin de tipo II reconocen secuencias de DNA especficas y cortan
fuera de esas secuencias. Cierto o Falso.
3. Mencione de que est hecho el gel de agarosa y el gel de poliacrilamida.
4. Mencione un ejemplo de una enzima que genere terminales cohesivos y una
que genere
terminales abruptos.
33
33
PRACTICA 9
SDS POLYACRYLAMIDE GEL ELECTROPHORESIS (SDS-PAGE)
Electrophoresis is the migration of charged molecules in solution in response to an electric field.
Their rate of migration depends on the strength of the field; on the nett charge, size and shape
of the molecules and also on the ionic strength, viscosity and temperature of the medium in
which the molecules are moving. As an analytical tool, electrophoresis is simple, rapid and highly
sensitive. It is used analytically to study the properties of a single charged species, and as a
separation technique.
Support Matrices
Generally the sample is run in a support matrix such as paper, cellulose acetate, starch gel,
agarose or polyacrylamide gel. The matrix inhibits convective mixing caused by heating and
provides a record of the electrophoretic run: at the end of the run, the matrix can be stained and
used for scanning, autoradiography or storage.
In addition, the most commonly used support matrices - agarose and polyacrylamide - provide a
means of separating molecules by size, in that they are porous gels. A porous gel may act as a
sieve by retarding, or in some cases completely obstructing, the movement of large
macromolecules while allowing smaller molecules to migrate freely. Because dilute agarose gels
are generally more rigid and easy to handle than polyacrylamide of the same concentration,
agarose is used to separate larger macromolecules such as nucleic acids, large proteins and
protein complexes. Polyacrylamide, which is easy to handle and to make at higher
concentrations, is used to separate most proteins and small oligonucleotides that require a small
gel pore size for retardation.
Separation of Proteins and Nucleic Acids
Proteins are amphoteric compounds; their nett charge therefore is determined by the pH of the
medium in which they are suspended. In a solution with a pH above its isoelectric point, a
protein has a nett negative charge and migrates towards the anode in an electrical field. Below
its isoelectric point, the protein is positively charged and migrates towards the cathode. The nett
charge carried by a protein is in addition independent of its size - ie: the charge carried per unit
mass (or length, given proteins and nucleic acids are linear macromolecules) of molecule differs
from protein to protein. At a given pH therefore, and under non-denaturing conditions, the
electrophoretic separation of proteins is determined by both size and charge of the molecules.
Nucleic acids however, remain negative at any pH used for electrophoresis and in addition carry
a fixed negative charge per unit length of molecule, provided by the PO4 group of each
nucleotide of the the nucleic acid. Electrophoretic separation of nucleic acids therefore is strictly
according to size.
SDS- PAGE OF PROTEINS
Separation of Proteins under Denaturing conditions
34
Sodium dodecyl sulphate (SDS) is an anionic detergent which denatures proteins by "wrapping
around" the polypeptide backbone - and SDS binds to proteins fairly specifically in a mass ratio
of 1.4:1. In so doing, SDS confers a negative charge to the polypeptide in proportion to its
length - ie: the denatured polypeptides become "rods" of negative charge cloud with equal
charge or charge densities per unit length. It is usually necessary to reduce disulphide bridges in
proteins before they adopt the random-coil configuration necessary for separation by size: this is
done with 2- mercaptoethanol or dithiothreitol. In denaturing SDS-PAGE separations
therefore, migration is determined not by intrinsic electrical charge of the polypeptide, but by
molecular weight.
PROTOCOLO
Preparacin de extractos de protena bacteriana y separacin sobre geles SDS-PAGE
12%, 0.8 mm, Sistema Laemmli.
35
Sample buffer
Vol = 13ml
Vol = 5 ml
Vol = 1,01 ml
H2O Destilada
3 ml
Sol 1
3,75 ml
Sol 2
6 ml
10% SDS
0.15 ml
Liberar O2 15min
APS 10%
75 l
TEMED
7,5 l
H2O destilada
Sol 3
Sol 2
10% SDS
APS 10%
TEMED
2,67 ml
1,25 ml
1,00 ml
0,05 ml
25 l
5 l
Sol 3
0,25 ml
Glicerol 50% 0,25 ml
10% SDS
0,40 ml
MET
0,10 ml
1% BPB
10 l
3 mA durante la noche.
o bien Voltios constantes: 50 V para hacer entrar las muestras en el gel.
150 V para la separacin.
5. Cuando el frente de migracin se encuentre en el borde abajo del gel, desmontar el equipo,
abrir las placas e introducir el gel en la solucin de coloracin, durante 15 minutos a 40C.
6. Lavar brevemente el gel con agua destilada y transferirlo en la solucin de decoloracin.
Agitar a 40C hasta decoloracin total. Cambiar de decolorante si es necesario (acompaar
de una esponja).
7. Interpretar los resultados.
36
Reactivos necesarios:
IMPORTANTE!: Usar guantes o mscara de proteccin para la manipulacin de sustancias
txicas
-PREPARAR LA CANTIDAD DESIGNADA EN NEGRILLAS500 ml Agua destilada
50 ml Sol 1L:
1.5 M Tris HCl pH 8.84 (18.15g de Tris en 28 ml de HCl1N, ajustar el pH a 8.8 a 20C con HCl
1 N. Completar a 100 ml.
50 ml Sol 2:
30g Archilamida + 0.8g Bisacrilamida, H2O a 100 ml. Filtrar, agregar una esptula de
Amberlita MB-1 o solucin Protogel (el pH debe superar la neutralidad).
50 ml Sol 3L:
0.5 M Tris- HCl 1 N, ajustar el pH a 6.8 a 20C con HCl 1N, completar a 25 ml.
2 litros Tampn de electroforesis: 50 mM de Tris, 384 mM Glicina, 0.1% de SDS pH 8.3 (30g
Tris, 144 g Glicina, 5 g SDS Na en 5 litros de agua destilada).
20 ml 10% SDS (50 ml)
1 ml 10% Persulfato de amonio (APS) guardar en congelador
TEMED (Stock)
2- Mercaptoetanol (MET) (Stock)
20 ml n-butanol
5 ml 50% glicerol
500 l 1% Azul de bromofenol (BPB)
500 ml Solucin de coloracin: 0.25% Coomassie blue, 50% metanol, 10% cido actico (2.5
g Coomassie blue R250, 500 ml de metanol, 100 ml de cido actico, 400 ml de agua
destilada).
2 litros Solucin de de-coloracin: 7% cido actico, 5% metanol (4400 ml de agua destilada,
350 ml de cido actico, 250 ml de metanol).
10 ml de STE buffer : (0.1 M NaCL,10 mM Tris HCL pH 8.0, 1 mM EDTA pH 8.0)
500 ml de Nitrgeno liquido
Materiales :
Pedazo de esponja (espuma de colchn)
Cubeta de plstico (p.ej. de cocina) largo = 25 cm, ancho 20 cm y alto 15 cm.
Guantes plsticos (suficientes)
Pipetas pasteur
Nuckies
Papel de filtro y embudo de vidrio
1 Beakers de 50 ml
Fuente de hielo picado
Equipos:
Kit de electroforesis de protenas
Bao a 100C (Puede ser beaker 500 ml sobre estufa)
Bao ultrasnico (sonicador) o bomba con campana de vaco.
Agitador de barras magnticas
Micropipetas
Estufa a 40C
Vrtex
Espectrofotmetro
Microcentrfuga
Material biolgico:
Precultivo de cepas bacterianas (cultivo de 20 ml medio LB).
37
38
BIBLIOGRAFIA
WEB
http://www.doaj.org
http://www.jstage.jst.go.jp/browse/
http://www.highwire.org
http://www.pnas.org
http://www1.embl-heidelberg.de/
http://au.expasy.org/
http://www.cellbio.com/
http://www.horizonpress.com/gateway/
http://www.ornl.gov/sci/techresources/Human_Genome/education/education.shtml
http://www.bioinformacion.net/tablabiologiamolecular.htm
http://www.biology.arizona.edu/molecular_bio/molecular_bio.html