I am looking for a protocol for DNA extraction from bucal swabs or saliva but most of them use kits and I only have reactives for traditional methods.
You can use phenol-chloroform isolation method, but the concentration of DNA will be low.
Because of the limitation of your material, if you use the traditional method to extract DNA, the quality and quantity may be low.
You can refer to this web page
http://bitesizebio.com/3651/practical-application-of-phenolchloroform-extraction/
Hope it useful to you!
Hi, How do you collect your sample?. you can get larger amount of cells buy gentle swapping or brushing of cheeks exfoliating cells with PBS. you can then centrifuge the sample and try a protocol with few steps to avoid loosing DNA also kits for tissue extraction will work for that sample.
I agree with Misti. 5% Chelex is cheap and easy. We use this for oral swab standards in my lab. You end up with a high elution volume, so you may potentially need to concentrate the samples a bit (speedvac). If you use chelex, transfer your final DNA sample off the chelex beads because they can start to mess with your DNA sample if the DNA is sitting on them for more than a few weeks.
Here is a rough version of my lab's protocol
1. Using a clean blade and cutting surface for each sample, dissect an appropriate amount
from the swab (~1/4 of the swab should be plenty) and place into a clean microcentrifuge tube.
2. Add to the substrate between 300 μL to 1 mL of autoclaved deionized water in a microcentrifuge tube.
3. Incubate at room temperature for at least 30 minutes to rehydrate the sample.
4. Twirl the swab or fabric substrate with a sterilized toothpick to release the cells. Remove
the substrate with the toothpick and "squeeze out" against side of tube. Alternatively, place the substrate into a spin basket and
proceed to the next step.
5. Spin the samples in a microcentrifuge for 2 - 3 minutes at maximum speed.
6. Without disturbing the pellet, remove all but approximately 50 μL of supernatant.
7. Add 5% Chelex to a final volume of 200 μL.
8. Add 2 μL of 18 mg/mL Proteinase K. Mix gently.
9. Incubate the samples at 56° C for 15 - 30 minutes.
10. Vortex the samples at high speed for 5 - 10 seconds.
11. Spin the samples in a microcentrifuge for 10 - 20 seconds at maximum speed at room
temperature.
12. Incubate the samples in a boiling water bath for 8 minutes.
13. Vortex the samples at high speed for 5 - 10 seconds.
14. Spin the samples in a microcentrifuge for 1-3 minutes at maximum speed.
15. Transfer sample to a new tube avoiding transfering the chelex beads.
16. To store the DNA extracts, refrigerate or freeze.
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