Fecha/hora: 16/06/2024 06:39:34

Biblioteca(s): | INIA Tacuarembó.
Fecha corriente: | 13/03/2018
Fecha de la última actualización: | 13/03/2018
Autoria: | NIKICHUK, N.; TORRES, D.
Afiliación: | NATALIA ISABEL NIKICHUK BELL, INIA (Instituto Nacional de Investigación Agropecuaria), Uruguay; DIEGO GABRIEL TORRES DINI, INIA (Instituto Nacional de Investigación Agropecuaria), Uruguay.
Título: | Protocol: fast and high-performance Eucalyptus DNA extraction in 96-well plate method.
Año de publicación: | 2014
Fuente/Imprenta: | In: IUFRO Forest Tree Breeding Conference, August 25-29, Prague, Czech Republic, 2014. Book of Abstracts.
Páginas: | p. 54
Idioma: | Inglés

Contenido:

Forestry genetics and genomics require a great number  of samples to produce accurate results. This is a simple method   with   high-throughput   DNA   extraction   that   combined  CTAB  protocol  with  fast  rupture  tissue  and  centrifuge  steps  in  plates  to  obtain  good  yield,  quality  and   low   price.   Approximately   576   samples   can   be   processed for day. The obtained DNA is suitable for use in  PCR-based  study  like  Microsatellites.  This  method  employs   96   Racked   Collection   Microtubes   (Qiagen).   Eucalyptus  tissue 15mg was placed in each well together with  a  tungsten  bead  and  420ul  of  CTAB2X  buffer  distributed  with  a  multichannel  pipette,  the  samples  were  disrupted  to  full  power  in  a  TissueLyser  (Qiagen)  for 3min. Then we added 360ul of CIA24:1 to each tube 
and both racks were centrifuged at 4000 RPM for 20min (ThermoScientific  SorvallST16).  Afterwards  the  upper  phases  were  transferred  carefully  to  clean  rack-wells  and  150ul  of  isopropanol  were  added  to  each  well  and  centrifuged  at  4000RPM  for  20min.  The  liquid  phases  were  removed  and  the  pellets  were  washed  twice  with  ethanol  70%  and  centrifuged  at  4000RPM  for  5min.  Finally the pellets were resuspended in 100ul of RNAse and  incubated  at  37ºC  for  60min.  To  evaluate  the  yield  and  purity,  this  prococol  was  compared  against  results obtained  with  four  commercial  extraction  kits:  DNeasy  Plant Handbook® (Qiagen), Power Plant DNA® Isolation Kit  (MOBIO),  Wizard®  Genomic  DNA  Purification  Kit  (Promega), ZR Plant / Seed DNA MiniPrep® (Zymo). The 
procedure of rupture was the same for all protocols and in  all  cases  racks  of  96  wells  were  used.  The  data  of  absorption  spectrum  indicate  an  average  concentration  of 400ng/ul for CTAB protocol, Qiagen 150ng/ul, Wizard 165ng/ul   MOBIO   85ng/ul   for   Zymo.   Comparing   this   method  to  the  commercial  is  demostrated  that  the  final  concentration  superior,  the  time  os  processing  is  lower,  also it is considerable cheaper.

Palabras-Clave: | GENÉTICA FORESTAL.
Thesagro: | FORESTACIÓN.
Categoría del tema: | K10 Producción forestal
URL: | http://www.ainfo.inia.uy/digital/bitstream/item/8912/1/Protocol-fast-and-high-performance-Eucalyptus-DNA.pdf
Registro original: | INIA Tacuarembó (TBO)
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