# **DNA extraction**
## Preparation:
| Material | Quantity per sample | Note |
| :----------: | :---: | :---: |
| DNA extraction buffer | 400 µL | See the last page |
| Isopropanol (2-propanol) | 300 µL | Around 70% of the supernatant |
| Ethanol (EtOH) | 1000 µL | DNase-free ethanol |
| Tris-EDTA | 100 µL | Commercial product |
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# Procedure
## Step 1: Cell lysis
> Caution:
If directly powderized the sample with liquid nitrogen within the ependorf, after filling in the liquid nitrogen into the eppendorf, DO NOT close the lid, the eppendorf will explode as temperature raise.
1. Put two steel balls into each 1.5 mL eppendorf;
> - The steel balls must had been washed with 70% EtOH and autoclaved.
2. Add 400 µL DNA extraction buffer into each eppendorf;
3. Directly submerge the samples into the DNA extraction buffer in each eppendorf;
4. Homogenize the samples and the buffer with a homogenizer;
> - There are two containers for the homogenizer;
> - each containers should be balanced (the amount and placement of the samples);
> - put a tissue on top of the eppendorfs' lid, this can prevent the solution seep out to the container bottom, and also eliminates the space between the container and the cover, improves grinding efficiency;
> - after mounted the containers on the machine, fasten tight the big roller, and stuck in the tenons.
5. Confirm the machine settings and start homogenizing;
> - Frequency : 30 hits / sec ( maximum frequency )
> - Time : 2.0 mins × 3
6. Centrifuge at room temperature ( > 15000 rpm, 20 mins ) to bring down the bubbles;
7. Dry bath ( 60°C, > 1 hour );
## Step 2: Disrupt protein—DNA interactions
8. Add 400 µL 5 M NaCl ( same volume with the extraction buffer, therefore the NaCl final concentration will be 2.5 M );
9. Centrifuge at room temperature ( > 15000 rpm, 20 mins ) to bring down the remaining tissues;
## Step 3: Collect DNA supernatant
10. Carefully take the supernatant ( around 90% of the supernatant volume ) to a new 1.5 mL eppendorf;
## Step 4: Precipitate DNA
11. Add 300 µL isopropanol ( around 70% of the supernatant volume ) into the supernatant, leave overnight at -20°C;
12. Centrifuge at 4°C ( > 15000 rpm, 20 mins ) to bring down the DNA;
13. Discard the supernatant carefully. Do not touch the tube bottom, which sticking with the DNA palette;
## Step 5: Wash
14. Add 200 µL 70% ethanol and gently invert the tube;
15. Centrifuge at 4°C ( > 15000 rpm, 20 mins ) to bring down the DNA;
16. Discard the ethanol carefully with small pipette;
## Step 6: Elute DNA
17. Dry bath at 60°C for around 10 mins, the ethanol should be totally gone;
18. Add 50 µL DNase-free water or TE buffer ( pH 8.0 ) and dry bath at 60°C for around 10 mins to dissolve the DNA palette;
19. After the DNA was fully dissolved, slightly centrifuge at room temperature to bring down all solution on the tube wall;
- Store the purified DNA at −20°C for up to 2 years. Avoid repeated freeze–thaw cycles and vigorous mixing.
## Step 7: DNA quality assurance:
20. Check the DNA concentration and quality.
- Reference: https://ntuhmc.ntuh.gov.tw/epaper-57th.htm
21. Standardize the DNA concentration according to the lowest concentration of the samples.
- At least 10 ng / µL for PCR
- the diluted DNA should be used as soon as possible
### Wavelength detection
| Wavelength (nm) | Detected Compound |
| :---: | :--- |
| 230 | EtOH, EDTA, carbohydrates, phenol, Guanidone HCL (for DNA isolation) |
| 260 | RNA,ssDNA, dsDNA, guanidine isothiocyabate (for RNA isolation) |
| 270 | Phenolic solution (TRIzol for RNA isolation) |
| 280 | Protein, phenol, other contamination |
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### Ideal wavelength ratio (quality indicator):
| Nucleic acid | 260 / 280 nm | 260 / 230 nm | 260 / 270 nm |
| :---: | :---: | :---: | :---: |
| DNA | ~ 1.8 | > 2.0 | > 1.2 |
| RNA | ~ 2.0 | > 2.0 | > 1.2 |
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Done !!!
# Stock solutions
## DNA extraction buffer
| Chemical | Concentration | Addition |
| :---------- | :---: | :---: |
| 1 M Tris-HCl (pH 7.5) ※ | 200 mM | 20 mL |
| 5 M NaCl | 250 mM | 5 mL |
| 500 mM EDTA † | 25 mM | 5 mL |
| 10% SDS § | 0.5% (v/v) | 5 mL |
| ddH2O | | 65 mL |
| Total: | | 100 mL |
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## Tris-HCl stock solution ( 1 M )
1. Dissolve 121.1 g of Tris base in 800 mL of ddH2O;
2. Adjust the pH to the desired value by adding concentrated HCl;
3. Allow the solution to cool to room temperature before making final adjustments to the pH;
4. Adjust the volume of the solution to 1 L with ddH2O. Dispense into aliquots and sterilize by autoclaving.
> | pH | 12N HCl addition |
> | :---: | :---: |
> | 7.4 | 70 mL |
> | 7.6 | 60 mL |
> | 8.0 | 42 mL |
> |||
## EDTA stock solution ( 0.5 M, pH 8.0 )
> EDTA ( Ethylenediamenetetraacetic acid )
1. Add 186.1 g of disodium EDTA • 2H2O to 800 mL of ddH2O;
2. Stir vigorously on a magnetic stirrer to dissolve the EDTA;
3. Adjust the pH to 8.0 with NaOH (~ 20 g of NaOH pellets);
> Note: The disodium salt of EDTA will not go into solution until the pH is adjusted to ~ 8.0 by the addition of NaOH.
4. Dispense into aliquots and sterilize by autoclaving.
## SDS stock solution ( 10% )
> Sodium dodecyl sulfate, sodium lauryl sulfate
1. Dissolve 100 g of electropheresis-grade SDS in 900 mL of ddH2O;
2. Heat to 68°C and stir with a magnetic stirrer to assist dissolution. If necessary, adjust the pH to 7.2 by adding a few drops of concentrated HCl;
3. Adjust the volume to 1 L with ddH2O;
4. Store at room temperature. Sterilization is not necessary.
DO NOT autoclave.