Excellgen at 18:07, 24 August 2016

2016-08-24T18:07:29Z

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'''Extraction Buffer A (EBA) Per 100 mL'''

{| style="width:500px" border="1" cellpadding="1" cellspacing="1"
|-
| 2% (w/v) hexadecyltrimethylammonium bromide (CTAB) 
| 2.0 g 
|-
| 100 mM Tris (pH 8.0), Use 1 M stock 
| 10 mL  
|-
| 20 mM EDTA (Use 0.5 M stock)  
| 1 mL  
|-
| 1.4 M NaCl 
| 8.2 g  
|-
| 4% (w/v) polyvinylpyrrolidone (PVP) mw:~4000 
| 4.0 g 
|-
| 0.1% (w/v) ascorbic acid , optional 
| 0.1 g 
|-
| 10 mM β-mercaptoethanol (BME)* (Use 14.3 M stock) 
| 70 µL 
|}

''' Extraction Buffer B (EBB) Per 100 mL '''

{| style="width:500px" border="1" cellpadding="1" cellspacing="1"
|-
| 50 mM EDTA (Use 0.5 M stock) 
| 2.5 mL  
|-
| 100 mM NaCl 
| 0.6 g 
|-
| 10 mM β-mercaptoethanol (BME)* (Use 14.3 M stock) 
| 70 µL 
|}

''' 10 mM Tris (pH 8.0) (Use 1 M stock)'''

{| style="width:500px" border="1" cellpadding="1" cellspacing="1"
|-
| 1.0 mL TE Buffer Per 
| 100 mL  
|-
| 1 mM EDTA (Use 0.5 M stock) 
| 50 µl 
|}

'''Other Required Reagents''' 5 M potassium acetate (Stored at –20oC) 3 M sodium acetate (pH 5.2) 70% ethanol (stored at -20oC) Absolute isopropanol (stored at -20oC)

'''1 M Tris pH 8.0'''

Dissolve 121.1 g of Tris base in 800 ml of H2O. Adjust pH to 8.0 by adding 42 ml of concentrated HCL.

'''Binding Buffer:''' (2 M Guanidinhydrochloride, 75 % (v/v) Ethanol)

or   24 g guanidinium thiocyanate GuSCN, 20 mL 0.1 M Tris-HCl, pH 6.4,,Heat to 60°C to dissolve the GuSCN., Add 4.4 mL 0.2 M EDTA, pH 8.0. Mix by inverting.

'''Column Washing Buffer I: '''(10 mM NaCl, 10 mM Tris-HCl pH 6.5, 80 % (v/v) Ethanol).

'''Column Washing Buffer II: '''(96 % Ethanol). 

'''Elution Buffer: '''

'''Extraction Protocol: precipitation using isopropanol''' 1. Weight out 0.3 g of plant tissue 2. Place tissue on a clean glass slide. Chop the tissue into a paste using a clean single edge razor blade. (we have also modified a Dremel Roto-tool for use as a simple tissue homogenizer with good success) 3. Immediately transfer tissue to a 1.5 mL microcentrifuge tube (use Kontes #749520-0090) and (optional) further grind tissue with a tube pestle (Kontes #749521-1590) 4. Once the sample is prepared add 400 µL EBA, <s>900µl EBB,</s>  and  100 µl SDS. 5. Vortex and incubate at 65o C for 30 min. 6. Place tube on ice and add 400 µL cold 5 M potassium acetate. Mix by inversion and place tube back on ice for 3 min. 7. Centrifuge at 13,200 rpm for 10 min. (If possible, use a refrigerated microcentrifuge set to 4oC) 8. Transfer  supernatant to a new 1.5 mL microcentrifuge tube, add 5 ul RNase A, incubate at 37 oC for 30 min, then add 500 µL of ice cold absolute isopropanol, and incubate in ice for 20 min. 9. Centrifuge at 10,200 rpm for 10 min. discard the supernatant. Wash the pellet once in 250 µL 90% ethanol and let dry 10. Resuspend the dry pellet in 50 µL of TE.

QC DNA by nanodrop

'''Extraction Protocol: purification using a spin colum'''

4. Once the sample is prepared add 400 µL EBA and  100 µl SDS. 5. Vortex and incubate at 65o C for 30 min.

6. Place tube on ice and add 400 µL cold 5 M potassium acetate. Mix by inversion and place tube back on ice for 3 min. 7. Centrifuge at 13,200 rpm for 10 min. (If possible, use a refrigerated microcentrifuge set to 4oC)

6. Transfer supernatant to another tube, add 5 ul RNase A, incubate at 37 oC for 30 min

7. Cool on ice and add 500 µL cold Binding Buffer. Mix by inversion and place tube back on ice for 3 min. 8. Load to a spin column. Spin

9. Wash column with </span250 ul '''Column '''Washing Buffer I, spin

'''10. '''Wash column with 250 ul '''Column '''Washing Buffer II, spin,

'''11.  '''Elute DNA with 50 ul Elution Buffer preheated to 65 oC''.''

''[[File:Genomic DNA extraction.jpg|RTENOTITLE]]''

'''1:precipitation using isopropanol'''

'''2:purification using a spin colum'''

''''''

== Protocol for NGS ==

=== Materials and methods ===

Consumables 50 mL Falcon Tubes

RNAse A (Sigma Cat No. R6513)

Polyvinylpyrrolidone (PVP) (Sigma Cat No. PVP10) (not required)

Chloroform: Isoamyl alcohol 24:1 (Sigma Cat No. C0549)

Liquid nitrogen

β-mercaptoethanol (Sigma Cat No. 63689)

Trizma base (Sigma Cat No. 1503)

Ethylenediaminetetraacetic acid disodium salt dihydrate (EDTA) (Chem Supply Cat No. EA023)

Agarose (Amresco Cat No. 0710)

Sodium Chloride (NaCl) (Ajax Finechem Cat No. 1103414)

Hexadecyltrimethylammonium bromide (CTAB) (Sigma Cat No. 52365)

Eco RI (not required- used for quality assurance) (NEB Cat No. R0101S)

Hin dIII-HF (not required- used for quality assurance) (NEB Cat No. R3104S)

=== Reagents ===

Extraction Buffer: 100 mM Tris-HCl (pH 7.5), 25 mM EDTA, 1.5 M NaCl, 2% (w/v) CTAB, and 0.3% (v/v) β-mercaptoethanol- added immediately before use

RNAse A stock solution (10 mg/mL)

5 M NaCl

95% ethanol (v/v)

70% ethanol (v/v)

TE Buffer: 10 mM Tris-HCl (pH 7.6), 0.1 mM EDTA

CutSmart Buffer (NEB Cat No. B7204S)

NEBuffer EcoRI (NEB Cat No. B0101S)

=== Equipment ===

Mortar and Pestle

Water Baths (65°C and 37°C)

Centrifuge (capable of spinning 50 mL centrifuge tubes at 5000 × g) 20°C Freezer Gel electrophoresis system (e.g. Jordan Scientific JP-250)

NanoDrop UV/Vis Spectrophotometer (e.g. NanoDrop 8000, Thermo Scientific)

=== Plant material and tissue collection ===

 Leaf tissue of Corymbia citriodora subsp. variegata, Corymbia henryi, Corymbia torelliana, and Corymbia citriodora subsp. citriodora was obtained from Queensland Department of Agriculture, Fisheries and Forestry in Gympie, Australia. Leaf tissue of Coffea brassii was obtained from the Australian Tropical Herbarium in Cairns, Australia. Leaf material after harvesting was transported on ice and stored at -80°C until subjected to DNA extraction.

=== Protocol ===

 '''Preparatory steps''' Before grinding, pre-chill the mortar and pestle (to minimize frozen tissue thawing) and 95% ethanol solution at -20°C. Pre-heat water baths (65°C and 37°C) before beginning the extraction. Once pre-heated, prepare 10 mL (per 1 g of leaf tissue) extraction buffer by adding 0.3% (v/v) β-mercaptoethanol in a 50 mL Falcon tube, and pre-heat in the 65°C water bath. PVP can also be added at this point, but is not required.

'''Grinding and tissue disruption''' Using liquid nitrogen, grind 1 g of frozen leaf tissue into a fine powder. Place the powder into a new 50 mL Falcon tube and mix in the pre-heated extraction buffer. Put the sample into the 65°C water bath and mix by inversion every 10 min for 30 min- 1 h. After incubation, centrifuge the sample tube for 5 min at 5000 × g (to pellet and remove un-lysed leaf tissue) and decant the supernatant into a new 50 mL Falcon tube.

'''Protein Extraction and RNAse treatment''' Add 1 volume of chloroform: isoamyl alcohol to the solution and mix by inversion for 5 min. Centrifuge the sample for 10 min at 5000 × g and pipette the upper aqueous phase into a new Falcon tube, taking care to avoid the aqueous/organic layer interface. Add 5 μL of RNAse A (10 mg/mL) to the solution and incubate at 37°C for 15 min with periodic, gentle mixing. After incubation, add 1 volume of chloroform: isoamyl alcohol to the solution and mix by inversion for 5 min. Centrifuge the solution for 10 min at 5000 × g and pipette the aqueous phase into a new Falcon tube, again taking care to avoid the organic layer.

'''Precipitation''' Add ½ volume of 5 M NaCl to the sample and mix gently by inversion. Then, add 3 volumes of cold 95% ethanol and mix gently by inversion. Place the tubes into a -20°C freezer and incubate for 1 h. NOTE: do not leave the sample at -20°C for more than 1 h as both the CTAB and NaCl can precipitate from solution, preventing DNA isolation.

After incubation, centrifuge the Falcon tube for 10 min at 5000 × g to pellet the DNA. Carefully decant away the supernatant and wash the DNA pellet with 3 mL of 70% ethanol. Gently swirl the solution and centrifuge again for 10 min at 5000 × g. Carefully decant the supernatant and air-dry DNA pellet for 15 min at room temperature. Once dried, suspend DNA in 200 μL of TE buffer.

'''DNA quality and quantity assessment''' Assess the quality of the extracted DNA using a NanoDrop UV/Vis spectrophotometer and 0.7% (w/v) agarose gel, looking for a single absorbance peak at 260 nm, a 260/280 absorbance ratio of 1.8-2.0, and no evidence of substantial band shearing or contamination (either RNA or polysaccharide).
[[Category:Protocols]]

Genomic DNA from Plants - Revision history

Excellgen at 18:07, 24 August 2016