Bacterial and Yeast Genomic DNA Extraction Protocol
Reagents:
[edit]Ethanol 96%
NaOH
EDTA
Silica column (QIAamp DNA mini kit, Qiagen)
Triton TM X-100
Tween-20
Nutrient broth
Solution
[edit]EtNa DNA extraction solution
| Component |
Volume (mL) |
Concentration in EtNa with bacteria extraction solution |
Concentration with bacteria |
| NaOH 2 M |
5.5 |
240 mM |
200 mM |
| Ethanol 96% |
35 |
74 % |
61% |
| EDTA 0.025 M |
5 |
2.7 mM |
2.25 mM |
| Volume |
45.5 |
This solution is stable for one month when stored at room temperature in a glass bottle.
| Component |
Volume (mL) |
Final |
| Tris-HCl, pH 8.0, 0.5 M |
5 |
50 mM |
| EDTA 0.5 M |
0.01 |
0.1 mM |
| Triton-x-100 |
0.5 |
1% |
| Tween20 |
0.25 |
0.5% |
| H2O |
44.25 |
|
| Volume |
50.01 |
This solution is stable for 1 year at 4°C.
Procedure
[edit]1. Begin by placing 100 μL of the sample in a microcentrifuge tube. This sample may be an overnight culture of a bacteria or yeast in nutrient broth, or bacterial or yeast colonies suspended in water, nutrient broth, or saline. Urine, cerebrospinal fluid, or plasma may also be used.
Tissue culture cells from one 24-well tissue culture dish scraped into 100 μL of DMEM
with 10% serum may also be used to extract cellular DNA or viral DNA.
2. Add 455 μL of EtNa DNA extraction reagent. Mix briefly.
3. Heat at 80°C for 10 min.
4. Spin at 16,060 × g for 10 min.
5. Remove the supernatant.
6. Suspend the pellet in 100 μL of DNA suspension solution.
EtNa pure
* In addition to the sample types for EtNa crude, a 10% fecal suspension (after allowing large particles to sediment) may also be used for EtNa pure.
7. Carefully apply the mixture from step 3 to the QIAamp mini spin column.
8. Close the cap and centrifuge at 6082 × g for 1 min.
9. Place the QIAamp Mini spin column in a clean tube and discard the tube containing the filtrate.
10. Carefully open the QIAamp Mini spin column and add 500 μL Buffer AW1. Close the cap and centrifuge at 6082 × g for 1 min. Place the QIAamp Mini spin column in a clean tube, and discard the collection tube containing the filtrate
11. Carefully open the QIAamp Mini spin column and add 500 μL Buffer AW2. Close the cap and centrifuge at 16,060 × g for 4 min. Place the QIAamp Mini spin column in a clean tube and discard the collection tube containing the filtrate.
12. Place the QIAamp Mini spin column in a clean 1.5-mL microcentrifuge tube and discard the collection tube containing the filtrate.
13. Carefully open the QIAamp Mini spin column and add 100 μL Buffer AE or distilled
water. Centrifuge at 6082 × g for 1 min