Promocionado por PROMEGA NL
Universidad Catolica de Louvain, Escuela de medicine (Bruselas, Belgium)


atelier de genetica molecular SBIM 2520
RNA extracción
regresa a la primera semana de trabajo

Maritza BARCIA MACAY* (Pharmacist), Daniel RUIZ** (Erasmus), Jean-Pierre HERVEG (Emeritus)***
*Universidad de Guayaquil, Ecuador, ** Universidad de Alcalá, Madrid, España, ***Universidad Catolica de Louvain, Belgica

extraction of the total RNA of the thyroid gland will be done with the promega kit Z3101 SV Total RNA Isolation System.

thyroid lobes are destroyed in a Potter homogenizer by a chaotropic agent, 8 M guanidium thiocyanate (lysis buffer), mechanical forces applied to the pestle and heat in a water bath. The lysate RNA and DNA are then be adsorbed on a resin, and cleaned by a wash buffer. DNA is destroyed by a DNAse acting on the resin. A DNAse stopping agent is added after 15 minutes to stop DNAse activity. RNA is washed again. Finally, RNA is washed out by RNAse free water.

 


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To the homogenizer containing the lysate add 350 ml of dilution buffer (blue liquid). Warm at 70° C for exactly 3 minutes.

centrifuge at 12000 g for 10 minutes.

transfer the clear supernatant (avoiding pellet debris) to a fresh microcentrifuge tube.


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prepare the spin column assembly.

add 200 ml of 95 % ethanol to the tube, mix by pipetting.

transfer this mixture to the spin column assembly.

centrifuge at 12000 g for 1 minutes

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remove the spin basket from the column assembly.

discard the liquid collected in the collection tube.

put the spin basket back on the empty collection tube.

add 600 µl of ethanol diluted wash solution to the spin column assembly.

centrifuge at 12000 g for 1 minutes.


was the wash solution diluted with ethanol?

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remove the spin basket from the column assembly.

discard the liquid collected in the collection tube.

in this order, pipet (by sample) multiples of 40 µl of core buffer, 5 µl of MnCl2 and 5 µl of DNAse. Mix.

pipet the DNAse mix so that it's yellow color covers the membrane of the pin basket.

incubate 15 minutes at 20-25° C.

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add 200 µl of DNAse stop solution.

centrifuge at 12000 g for 1 minutes.

It's not necessary to empty the collection tube right now.

add 600 µl of RNA wash solution.

centrifuge at 12000 g for 1 minutes and empty the collection tube.

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add 250 µl of RNA wash solution.

centrifuge at 12000 for two minutes.

remove the cap of the spin basket.

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replace the collection tube by an elution tube (in the packaging).

spread 100 µl of RNAse free water so that it covers the membrane of the spin basket.

place the elution/spin basket in the centrifuge, caps of the elution tube facing out.

centrifuge at 12000 for one minute.


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Preguntas

1. What do you know about chaotropic agents. How do they work?
2. What kind of a resin do they use to bind DNA?

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Promocionado por PROMEGA NL
Universidad Catolica de Louvain, Escuela de medicine (Bruselas, Belgium)


atelier de genetica molecular SBIM 2520
RNA extracción
regresa a la primera semana de trabajo

Maritza BARCIA MACAY* (Pharmacist), Daniel RUIZ** (Erasmus), Jean-Pierre HERVEG (Emeritus)***
*Universidad de Guayaquil, Ecuador, ** Universidad de Alcalá, Madrid, España, ***Universidad Catolica de Louvain, Belgica