DNA isolation protocol from plants

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DNA isolation protocol from plants

 INTRODUCTION :

Plant materials are among the most difficult for high quality DNA extractions. The key is

to properly prepare the tissues for extraction. In most cases this involves THE use of liquid nitrogen flash freezing followed by grinding the frozen tissue WITH a mortar and  pestle. Liquid nitrogen is difficult to handle and it is dangerous in an open laboratory  environment such as a classroom. For this reason we modified a very simple plant  DNA extraction protocol to use fresh tissue. We have also used tissue prepared in  advance by dessication. The protocols and results are presented here. 

The basic principle of DNA isolation is disruption of the cell wall, cell membrane, and nuclear membrane to release the highly intact DNA into solution followed by precipitation of DNA and removal of the contaminating biomolecules such as the proteins, polysaccharides, lipids, phenols, and other secondary metabolites. 

REAGENT AND BUFFERS :

The method involves extraction of DNA using a buffer (pH 8.0) containing Tris (100 mM), EDTA (20 mM), 7 M urea, 0.5 M NaCl and 0.1% β-mercaptoethanol, followed by purification of DNA with phenol, chloroform and Isoamlyalcohol and finally precipitation of DNA by sodium acetate and isopropanol

 

Extraction Buffer A (EBA) Per 100 mL

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) 4.0 g

 0.1% (w/v) ascorbic acid 0.1 g

 10 mM β-mercaptoethanol (BME)* (Use 14.3 M stock) 70 µL

_________________________________________

Extraction Buffer B (EBB) Per 100 mL

100 mM Tris-HCl (pH 8.0) (Use 1 M stock) 10 mL 

 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

 _____________________________________________

TE Buffer Per 100 mL

10 mM Tris (pH 8.0) (Use 1 M stock) 1.0 mL 

 1 mM EDTA (Use 0.5 M stock) 50 µL

 20% (w/v) sodium dodecyl sulphate (SDS)

Other Required Reagents

 5 M potassium acetate (Stored at –20o

C)

 3 M sodium acetate (pH 5.2)

 70% ethanol (stored at -20o

C)

 Absolute isopropanol (stored at -20o

C)

EXTRACTION PROTOCOL :

Plant samples can be prepared by cryogenically grinding tissue in a mortar and pestle after chilling in liquid nitrogen. Freeze dried plants can be ground at room temperature. In either case, a fine powder is best for extracting DNA. For each 100 mg homogenized tissue use 500 µl of CTAB Extraction Buffer

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 300 µL EBA, 900µl EBB, and 100 µl SDS.

5. Vortex and incubate at 65o

C for 10 min.

6. Place tube on ice and add 410 µL cold potassium acetate. Mix by inversion and

place tube back on ice for 3 min.

7. Centrifuge at 13,200 rpm for 15 min. (If possible, use a refrigerated micro-

centrifuge set to 4o

C)

8. Transfer 1 mL of the supernatant to a new 1.5 mL microcentrifuge tube, add 540

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 500 µL 70% ethanol and let dry

10. Resuspend the dry pellet in 600 µL of TE. Add 60 µL 3M sodium acetate (pH 5.2)

and 360 µL ice cold absolute isopropanol. Incubate on ice for 20 min.

11. Repeat Steps 9−11 twice.

12. Resuspend the pellet in 50 µL TE and carry out agarose gel QC.

Agarose Gel QC :

1. Cast a 1.0% (w/v) regular agarose gel in 1X TBE

2. Place 5 µL of extracted DNA and 5 µL sterile water

 in a 0.2 mL microcentrifuge tube along with 2 µL of

 gel tracking dye.

3. Run the gel for 20 min. at 100v.

4. Stain gel and view result.

PCR QC :

Obtaining what appears to be good high molecular weight genomic is only the first line

of QC for this protocol. The ultimate test is to see if the DNA can be used to amplify a PCR product. The test case used in developing this protocol was leaf tissue from the coleus plant (Coleus blumei). In order to test the DNA for PCR amplification the gene encoding tyrosine aminotransferase (GenBank #AJ458993) was submitted for PCR primer selection using the IDT SciTools software PrimerQuest. The software chose the 

following sequences:

 Tat FOR:5’- ATA AAC CCT GGG AAC CCA TGT GGA –3’

 Tat REV: 5’- AAC TTT GGG CTC ATC AAA GTG CCG –3’

These sequences were synthesized and a PCR amplification carried out using the 

conditions; 5 min. 94o

C; 35 x (30 sec. 94oC, 30 sec. 

57oC; 30 sec 

72oC); 7 min.

                                                          🔗   END

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DNA isolation protocol from plants