DNA extraction is a physical and chemical method of DNA purification from cell membranes and other components including Proteins and RNA in addition to other cellular components.
Procedure:
There is a comprehensive and step-by-step protocol for DNA extraction given for researchers to understand and perform without any other assistance.
CTAB preparation
First of all, we need to prepare a CTAB solution in our laboratory to carry out DNA isolation as given below;
CTAB Buffer
- 100ml 1M Tris HCl pH 8.0
- 280ml 5 M NaCl
- 40ml of 0.5 M EDTA
- 20 g of CTAB (cetyltrimethylammonium bromide)
- Bring total volume to 1 L with ddH2O
TE Buffer
- 10ml 1 M Tris HCl pH 8.0
- 2ml 0.5 M EDTA
- Bring total volume to 1 L with ddH2O
1 M Tris HCl
- 121.1 g Tris dissolved in about 700 ml of H2O.
- Bring pH down to 8.0 by adding concentrated HCl (you’ll need about 50 ml).
- Bring total volume to 1 L with ddH2O.
0.5 M EDTA
- 186.12 g EDTA
- Add about 700 ml H2O
- 16-18 g of NaOH pellets
- Adjust pH to 8.0 with a few more pellets, EDTA won’t dissolve until the pH is near 8.0
- Bring total volume to 1 L with ddH2O.
5 M NaCl
- 292.2 g of NaCl
- 700 ml H2O
- Dissolve (don’t add NaCl all at once, it will never go into the solution) and bring to 1 L
7.5 M Ammonium acetate
- 57.81g ammonium acetate
- ~50 ml of H2O
- Bring to 100 ml total volume
After CTAB preparation simply follow the steps and get purified DNA in less time and effort as given;
1. Sample preparation
- Weigh the sample plant leaf up to 100mg in Tissue Lyser or pestle and mortar
2. Lysis
- Add 400ul 2% CTAB buffer.
3. Protein and RNA removal
- Add 10μl proteinase K and mix well by inverting
- Add 10μl RNase A solution to the lysis solution
- Incubate at 650 for 15-30 min (invert the tube every 5 min)
- Centrifuge for 5 min at 13000rpm
- Pick the supernatant 200ul of clean lysate to a new tube
4. DNA binding
- Add 650μl of binding buffer and invert to mix well
- Transfer the sample to the column tube of the gene jet
- Centrifuge for 1 min at 13000rpm
- Discard the flow through
5. Washing
- Add 500μl washing buffer
- Centrifuge for 1 min at 13000rpm
- Discard the flow through
- Centrifuge the column for an additional 1ml at 12000rpm
- Discard the flow through
- Transfer the sample to a new column tube of the gene jet
6. DNA Elution
- Add 50μl of elution buffer
- Collect DNA sample in a collecting tube and store them at 200C
Benefits
Some of the prominent benefits of the CTAB-based DNA extraction method are as given;
- Time-saving
- Cost-effective
- High yield of DNA
- High quality of DNA
