Extraction of DNA from a sample

Last Thursday, we had our first lab assignment, which was to extract our own DNA from a salivary sample and use the extracted DNA as PCR template. The following is the bench protocol followed by us. All the reagents used were from Qiagen, and the basic protocol was adapted from Qiagen's DNeasy Blood and Tissue Handbook.

Preparation:

1. Ensure that ethanol has been added to both the washing buffers (AW1 and AW2).
   
2. Set up a water bath at 56° Celsius.
   
3. Check to make sure that there is no precipitation in the Lysing buffer (AL). If there is any, re-dissolve it.
   
4. All the subsequent steps are preferably carried out at temperatures between 15° and 25° Celsius.
   

Procedure:

1. Trying to be as dignified as possible, collect some saliva in a 1.5 or 2 ml microcentrifuge tube. (Ideally, sample should come to about the 0.5 mark)
   
2. Centrifuge at 300 rpm for 5 to 10 minutes or till a firm pellet is formed at the bottom of the microcentrifuge tube.
   
3. Carefully pipet out the supernatant. This can be a tricky process as the pellet is easily dislodged. If the pellet is dislodged, re-centrifuge for 3 minutes.
   
4. Re-suspend pellet in 200μl PBS (Phosphate buffered Saline)
   
5. Add 20μl of proteinase K.
   
6. Add 200μl of buffer AL (Lysing buffer). Make sure that you are not using ATL or tissue lysing buffer at this stage. ATL is much stronger than AL.
   
7. Mix by vortexing, and incubate at 56° Celsius for 10 minutes.
   
8. Add 200μl ethanol (96% to 100%). Mix by vortexing.
   
9. Transfer the mixture into a DNeasy MiniSpin Column in a 2ml collection tube.
   
10. Centrifuge at 8000 rpm for 1 minute.
    
11. Discard liquid collected in the collection tube. If you have lots of collection tubes, you can discard the collection tubes also.
    
12. Place spin column in the emptied or a new 2 ml collection tube.
    
13. Add 500μl of buffer AW1 (Washing buffer 1).
    
14. Centrifuge at 8000 rpm for 1 minute.
    
15. Discard liquid in the collection tube. Changing collection tubes at this stage is once again optional.
    
16. Place spin column in the emptied or a new 2 ml collection tube.
    
17. Add 500μl of buffer AW2 (Washing buffer 2).
    
18. Centrifuge at 14,000 rpm for 3 minutes.
    
19. Remove the spin column carefully so it does not touch the flow through liquid or the collection tube.
    
20. You can discard the collection tube and the flow through at this stage.
    
21. Place the spin column in a new 1.5 or 2 ml microcentrifuge tube.
    
22. Add 200μl of the AE buffer (Elution buffer) to the spin column.
    
23. Incubate at room temperature for one minute.
    
24. Centrifuge at 6000 rpm for 1 minute.
    
25. If you want greater yield, repeat steps 21 to 24 again and combine the eluents from both the microcentrifuge tubes.
    

Conclusion:

• The DNA collected finally in the microcentrifuge can be used as the template for PCR experiments. Add 1μl of obtained eluent to 20μl of master mix and place in a thermocycler.