Miniprep Protocol for High Quality DNA Extraction from Plants
Author: Gayacharan

DNA isolation is a basic requirement for any molecular lab to perform molecular studies. Most of the DNA extraction and purification methods are developed aiming at large quantity DNA requirement, but many a times we require small quantity DNA but for large number of samples. In such cases we require cost effective method to extract enough quantity of DNA without compromising DNA quality. This not only reduce the extraction cost, but also requires less time. This method of DNA extraction may be highly useful to those molecular biologists working areas like marker assisted breeding, diversity analysis, germplasm screening, etc. Here we have discussed principles and method of DNA extraction.

Principles and method

A. Chemicals and solutions :

Several chemicals are required in different forms which are discussed below. Some are made as stock solutions for longer use and some are ready made available.

(1) 1M Tris solution:

Tris (hydroxymethyl) aminomethane ((HOCH2)3CNH 2, FW 121.14 g/mol) is a buffering agent extensively used in making buffer solution for DNA extraction (as discussed below), gel electrophoresis (TBE and TAE). For making 1M Tris-buffer solution, add molecular weight (121.4) g in one liter of milli-Q or double distilled water and adjust pH to 7.5 using HCl. The quantity of the buffer may be reduced or increased based on the scale of the work. The tris buffer has an effective pH range between 7.5 and 9.0. This buffer also inhibits number of enzymes, therefore should be taken care while studying proteins.

(2) 0.5M EDTA

Ethylene diamine tetra acetic acid ([CH2N(CH2CO 2H)2]2; FW 292.24 g/mol) is mainly used as chelating agent, i.e., its ability to sequester metal ions such as Ca 2+ and Fe3+ to reduce their reactivity with other compounds. It inactivates enzymes like DNase which uses metal ions (eg. Mg 2+) and keeps DNA safe from degradation. EDTA is mainly produced as disodium EDTA (C10H14N2Na2O 8•2H2O; FW 372.2 g/mol) and calcium disodium EDTA (C10H12N2O8CaNa2•2H2O; FW 374.27 g/mol) salts. To make 0.5 M EDT dissolve 18.6 g in 100ml milli-Q water and adjust pH 8.0 using NaOH. Note that EDTA salt does not dissolve in water until pH reaches to 8.0. Sometimes vigorous stirring with moderate heating is done.

(3) TE (Tris-EDTA) buffer

TE buffer is a commonly used in molecular biology to preserve DNA, cDNA or RNA in solution form while protecting from degradation. To prepare TE buffer mix 10 ml of 1M Tris (pH 7.5) and 2 ml of 0.5M of EDTA (pH 8.0).

(4) 10 % CetylTrimethyl Ammonium Bromide (CTAB)

CTAB ([(C16H33)N(CH3)3]Br, FW 364.45 g/mol) is a cationic detergent used in the DNA extraction buffer to remove membrane lipids and causes cell lysis. CTAB binds to the polysaccharides in high salt concentration condition (generally NaCl is used for this purpose), thus it removes polysaccharides from solution. Slowly add 10 g CTAB in milli-Q or double distilled water and stir with heat to dissolve and make up the volume to 100 ml.

(5) Plant DNA extraction buffer

Based on the scale of the work quantity of the DNA extraction can be made. Here it is given for 100 ml solution.

Component Quantity
10% CetylTrimethyl Ammonium Bromide (CTAB) 20 ml
10% PVP 20 ml
De-ionized water 9 ml
1M Tris-Cl 10 ml
0.5M Ethylene diamine tetra acetic acid (EDTA) 4 ml
4M NaCl 35 ml
2-mercaptoethanol (Freshly added) 2 ml
Total 100 ml


(6) Liquid nitrogen

Liquid nitrogen (LN) which has -196° C temperature is used for crushing leaf/tissues to make fine powder with minimal shearing of genetic material. Crushing leaf/tissues using LN has many positive roles like, it stops all cellular enzymatic activities, least DNA degradation, easy to crush tissues, evaporates water molecules from tissues, etc., thus it helps in obtaining good quality DNA or RNA.

(7) β-mercaptoethanol

β-mercaptoethanol or 2-Mercaptoethanol (HOCH2CH2 SH) is an reducing agent which helps in denaturing proteins by breaking the disulfide bonds (S-S bonds) between the cysteine amino acid residues. This results in disruption of both the tertiary structure and the quaternary structure of proteins. They also disrupt enzymatic activity of enzymes like ribonucleases, so prevents RNA degradation. It also helps in removing tannins and polyphenols. It is generally freshly added in DNA extraction buffer just before using DNA extraction buffer. Its half-life is more than 100 hours at pH 6.5 and 4 hours at pH 8.5.

(8) PVP ( Polyvinylpyrrollidone)

Polyphenols binds the DNA which alters the quality of results obtained from PCR and restriction digestion, and other uses. Further its oxidized product i.e. Quinone’s breaks the DNA by producing reactive oxygen species. PVP removes phenol compounds by forming hydrogen bonds with them, thus prevents polyphenols oxidation and browning of isolated DNA. β-mercaptoethanol, being strong reducing/antioxidant agent keeps polyphenols in reduced form.

(9) Phenol:Chloroform:Isoamyl alcohol

Phenol is a non-polar compound and helps in separating leaf tissue extract in aqueous and non-aqueous phases. Phenol in aqueous phase also helps in denaturing proteins. The aqueous phase is on top because it is less dense than the organic phase (phenol:chloroform). The proteins and hydrophobic lipids will partition into the lower organic phase while the nucleic acids (as well as other contaminants such as salts, sugars, etc.) remain in the upper aqueous phase. Chloroform and phenol mix well together, unlike phenol and water. The density of chloroform is 1.47 g/cm3 which is higher than that of water (1.0 g/cm3) and phenol (1.07 g/cm³). Mixing chloroform and phenol creates a denser solution than phenol alone, and therefore the separation of the organic compounds from the aqueous phase is even clearer than if only phenol is added in the tube. Isoamyl alcohol is added in small proportion to reduce foaming generated by chloroform and stabilize interphase (coagulated proteins). It also inhibits RNase activity and prevents the solubilization of long RNA molecules in the phenol phase with long poly-A tail.

(10) Ice cold isopropanol or absolute ethanol

For precipitation of DNA or RNA certain conditions are required: as salt (Nacl) to neutralize the charge on the nucleic acid backbone, making it less hydrophilic and fall out of solution; second is lower temperature to promote the flocculation of the nucleic acids so it readily pellets under the centrifugal force; third is nucleic concentration should be high enough to precipitate. As DNA is lesser soluble in isopropanol and ethanol, it precipitates. When only isopropanol is used in chlled condition, it precipitates DNA even in lower DNA concentration and salt also precipitates down. Other hand chilled ethanol precipitates DNA in high concentration condition but salt remain in solution. In general, DNA precipitates down in 35% isopropanol and 0.5 M salt or around final 75% ethanol concentration with 0.5 M salt.

B. DNA isolation steps:

  1. Crush approx. 0.5 g leaf/tissue sample in LN and add the leaf powder (a pinch) into Eppendorf tubes (2ml). For large scale DNA isolation, use tissue lyser machines e.g. QIAGEN TissueLyser LT. If this machine is used, based on its adapter capacity (generally 24 tubes/adapter), 2 ml safe lock tubes are used. Leaf samples are along with a steel ball is placed in each tube. Then tubes along with adapter are kept in LN for 2-3 mins for freezing of tissues and then immediately mounted on the machine for crushing with required vibration frequency. Alternatively DNA extraction buffer (500 µl) can be added and without freezing tissues can be crushed, but it comparatively yields poor quality DNA. For obtaining good quality of DNA, leaf samples should be taken before sunrise, and most tender tissues should be taken e.g. in rice, if plants are in plenty number, top central leaf should be pulled out slowly and inner most white tissues should be taken for DNA extraction. Tissues should be collected in LN box to avoid any enzymatic activities.
  2. Add 500 µl of DNA extraction (preheated at 65° C) buffer and 25µl β-mercaptoethenol in each tube containing crushed leaf tissues powder before it get any moisture. In case, if LN was not used in crushing, only 25µl β-mercaptoethenol is added in this step.
  3. Mix well and keep tubes in the hot water bath at 65°C for 1hr and alternatively hot air bath also can be used.
  4. Add equal volume (500 µl) of Phenol:Chloroform: Isoamyl alcohol (25:24:1) to each tube.
  5. Shake gently for 15 mins, or automated shaker can be used.
  6. After the incubation centrifuge at 12000 rpm for 10 mins.
  7. Add equal volume (500µl) of Chloroform: Isoamyl alcohol (24:1) and shake gently for 15 mins and centrifuged at 12000 rpm for 10 mins.
  8. Take out supernatant and add three times (1500 µl) ice-cold absolute ethanol and mix well for few seconds.
  9. Incubate tubes for overnight at -20°C and next day centrifuge for 10 mins at 12000 rpm.
  10. Decant the ethanol and wash the DNA pellets in the lower bottom of tubes with 70 % ethanol for three times. (Each time 200-300 µl of ethanol filled in tubes and incubate for 5mins and centrifuge at 12000 rpm for 5 mins).
  11. After washing the DNA pellet evaporate the ethanol by placing opened lid.
  12. If highly purified DNA is required, add 200 µl RNase buffer or double distilled water and add 5 µl of DNase free RNase A to it and incubate at 37°C for 1 hr. Further repeat step 8 to 12.
  13. Dissolve the dried pellet of DNA in 100 µl of T10E 1 buffer (10 ml 1M Tris-Cl pH 7.5 + 2 ml 500mM EDTA pH 8.0) and keep at 4°C for 2-3 days and then at - 20°C for long time preservation.
Make working DNA samples for PCR or any other molecular work by quantifying DNA in 0.8 % agarose gel. For more accurate dilution take absorbance of samples at 260 nm in a spectrophotometer. In case of nanodrop spectrophotometer which requires 2 µl of samples, take reading at 260nm wavelength (for DNA). First calibrate with DNA dilution medium (TE buffer or water). 1 opitcal density (O.D.) unit absorbance reading equals to DNA concentration of 50 µg/ml. For getting more accuracy in DNA dilution, the final DNA concentration should not reach beyond 3 µg/ml. For understanding level of contamination of protein and RNA reading at 280 nm is also taken and A260/A280 ratio is obtained. For good quality DNA ratio should be between 1.8 and 2; higher than 2 indicates contamination of RNA and lower than 1.8 indicates contamination by proteins.

References:

1. Doyle, Jeff J. "Isolation of plant DNA from fresh tissue." Focus 12 (1990): 13-15.

2. Dellaporta, S. L., Wood, J., & Hicks, J. B. (1983). A plant DNA minipreparation: version II. Plant molecular biology reporter, 1(4), 19-21.

3. Saghai-Maroof, M. A., Soliman, K. M., Jorgensen, R. A., & Allard, R. W. (1984). Ribosomal DNA spacer-length polymorphisms in barley: Mendelian inheritance, chromosomal location, and population dynamics. PNAS, 81(24), 8014-8018.


About Author / Additional Info:
Scientist (Agri-biotechnology)