Apr 15, 2020

Public workspaceAcid nucleic extraction from rice dried leaves

PLOS One
DOI
dx.doi.org/10.17504/protocols.io.bcntiven
  • 1IRD, Montpellier, France
Martine Bangratz
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DOI: dx.doi.org/10.17504/protocols.io.bcntiven
External link: https://doi.org/10.1371/journal.pone.0232115
Protocol Citation: Martine Bangratz, Charlotte Tollenaere 2020. Acid nucleic extraction from rice dried leaves . protocols.io https://dx.doi.org/10.17504/protocols.io.bcntiven
Manuscript citation:
M. Bangratz, I. Wonni, K. Kini, M. Sondo, C. Brugidou, G. Béna, F. Gnacko, M. Barro, R. Koebnik, D. Silué, C. Tollenaere (2020) Design of a new multiplex PCR assay for rice pathogenic bacteria detection and its application to infer disease incidence and detect co-infection in rice fields in Burkina Faso. PLoS One.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 18, 2020
Last Modified: April 15, 2020
Protocol Integer ID: 33203
Abstract
Acid nucleic extraction of rice dried leaves by a CTAB method adapted from Li et al 2008.

Li R, Mock R, Huang Q, Abad J, Hartung J, Kinard G. A reliable and inexpensive method of nucleic acid extraction for the PCR-based detection of diverse plant pathogens. Journal of Virological Methods. 2008;154(1):48-55. doi: 10.1016/j.jviromet.2008.09.008.
Materials
MATERIALS
ReagentTE buffer
ReagentIsopropanol
ReagentNaClMerck MilliporeSigma (Sigma-Aldrich)Catalog #S-3014
ReagentHexadecyltrimethylammonium bromide (CTAB)Merck MilliporeSigma (Sigma-Aldrich)Catalog #H9151
Reagent1 M Tris/HCl Stock Solution (dissolved Tris base adjusted to pH 8.0 with HCl)
ReagentChloroform: Isoamyl Alcohol (24:1)
ReagentPolyvinylpyrrolidoneMerck MilliporeSigma (Sigma-Aldrich)Catalog #PVP40
Reagentethanol
Reagentsodium bisulfite Merck MilliporeSigma (Sigma-Aldrich)
ReagentNa2EDTA
Waterbath at 65°C
Qiagen TissueLyser II
Microcentrifuge at 4°C
Safety warnings
working with chloroform : Isoamyl alcohol under a fume hood
Before start
- Put 20-50 mg of dried rice leaves sample into a safe lock tube 2.0 ml containing two stainless steel beads, 5mm.

- Prepare CTAB Extraction Buffer (warm up the buffer for the CTAB dissolution) :
For 100 ml
2 g CTAB (2% w/v)
2 g PVP-40
10 ml 1M Tris-HCl, pH8.0
8.18 g NaCl
744,48 mg EDTA
0,5 g sodium bisulfite (add just before to use)
qs 100 ml H20


Grind the dried leaves with the Qiagen TissueLyser II until obtaining a fine powder
Add 1 ml CTAB extraction buffer (see 'before start' for the buffer content) and homogenize by vortexing.



Incubate at 65°C for 30 min. Periodically, mix gently the tubes during the incubation.Temperature65 °C Duration00:30:00

Centrifigation10000 x g, 4°C, 00:10:00

Transfer the supernatant (650µl) to a 1.5 ml microcentrifuge tube.

Add equal volume of chloroform/isoamyl alcohol (24:1)
After shaking, Centrifigation15000 x g, 4°C, 00:10:00

Transfer the supernatant (500µl) in a new 1.5 ml tube containing 350 µl of isopropanol (pre-chilled at -20°C).
Mix gently
Temperature-20 °C Duration00:30:00

Centrifigation15000 x g, 4°C, 00:10:00

Discard the supernatant . Wash the pellet with 70% ethanol.

Centrifigation15000 x g, 00:05:00

Remove the ethanol and air-dried the pellet.
Dissolve the pellet in 50 µl sterile water or TE buffer and conserve the DNA at -20°C.