You will use 1.5 mL tubes to extract the DNA samples from saliva.
To start, prepare each tube by labelling them with a permanent marker.
Even if you only have one sample, it’s good practice to label the tube clearly. For example, if the sample is from a person, you could use their initials. It’s also a good idea to mark the date of the sample.
Prepare Saline Solution
You will need salt water (saline solution) as a mouthwash to collect your cheek cells.
In a small glass or similar, mix a pinch of table salt with water. A shot glass is perfect for this. The measurements do not need to be exact. A pinch of salt for a small sip of water is a good rule of thumb.
You do not need to make much – a small sip is enough.
Why the salt water? In this protocol, the aim is to get a sample of DNA from cheek cells. Your saliva, after rinsing your mouth will naturally contain cheek cells, which will be broken open during the protocol to release the DNA. The salt, i.e. sodium chloride, is used to stabilise the DNA, once it has been released.
Rinsing your mouth
Pour the salt water into your mouth. Don’t use too much, just a small sip is enough. Rinse your inner cheeks vigorously for 30-60 seconds. When you are done, spit the saline solution into a new glass. (or, if the shot glass you used to mix the salt water is empty, you can reuse it).
The goal of this step is to loosen as many cells from your mouth as possible. You can use your teeth to gently scrape your cheeks and tongue while you are swirling the salt water around in your mouth. You can also touch your inner cheeks with your tongue. Careful to not hurt yourself – there’s no need for blood, just saliva with lots of cheek cells.
Transfer your sample into the microcentrifuge tube
For this step, you will use your saliva sample (1), the microcentrifuge tube you labelled in the beginning (2), and a transfer pipette (3).
Use the transfer pipette to transfer your saliva sample into the microcentrifuge tube. Fill it up to the 1.5 mL mark.
It is time to use the centrifuge. This will use gravitational force to concentrate the sample.
Put the centrifuge tube with your saliva sample into the centrifuge. Make sure to balance the centrifuge with another sample or with another counter weight.
If you only have one sample, the easiest way to balance the centrifuge is to fill another tube with water and use it as a balancing tube.
Using the centrifuge in an unbalanced way is dangerous and will break the device. Follow our tips for balancing a centrifuge in the manual here. In this case, for example, you could either use a second sample as a counter balance or fill up another tube with water. Tubes must always be balanced with another tube of equal weight.
Once the sample tube is balanced in the centrifuge rotor, close the lid and activate the centrifuge module. Set the speed to 4,000G and spin for 90 seconds.
Recovering the Pellet
Check the sample tube after centrifugation has finished. All the cheek cells should now be concentrated in a small white ball at the bottom of the tube (1). This is called a pellet. The remaining liquid (2), called the supernatant, should be clear.
In this step, you will remove the supernatant, so only the white pellet remains. Check that your pellet is firmly attached to the bottom of the tube. If it is, you can carefully pour the supernatant away.
If the pellet is not firmly attached to the bottom of the tube, try spinning the sample again in the centrifuge to attach it to the bottom of the tube. If it remains loose, you can use the micropipette with a fresh tip to slowly transfer the supernatant out of the tube. You can also try using the transfer pipette you used earlier, but it might be difficult to control and could end up disturbing the pellet.
Resuspending the Pellet
You should now have a white pellet in your sample tube. It should be about the size of a matchstick head.
If your pellet is smaller than a matchstick head, you may not enough cheek cells to get a concentrated DNA sample. In that case, go back to step 2 to concentrate additional cheek cells from saliva. You can use the same sample tube and simply add more sample to the existing pellet.
Once you have a large enough pellet, you can resuspend the cells into the remaining liquid that is still in the tube. You now have a concentrated cell sample in a small volume of liquid. Make sure the tube is closed, then mix the cells from the pellet into the liquid by flicking the tube. The cells of the pellet are now resuspended in the liquid.
In this next step, you will use the micropipette (1) to transfer the resuspended sample (2) into a 0.2mL PCR tube (3), so that you can heat it in the thermocycler.
First, set the adjustable pipette to the maximum volume of 20μl.
Make sure the pipette has a new pipette tip. Then use the pipette to transfer the cell mixture of the sample to the 0.2mL PCR tube. Carry on until the PCR tube is almost full, or until you have no sample left. Add as much of the cell mixture as possible to the PCR tube.
Labelling the PCR Tube
Finally, click the lid of the PCR tube closed and label the tube to identify the sample, similarly to the centrifuge tube.
Label the side of PCR tubes, not the lid. The PCR machine has a heated lid, so any ink on the tube lid might come off.
Heating the sample
In this step, you will use the thermocycler as a heat block to boil the cells and burst them open, to release the DNA into the solution.
Place your PCR tube with your sample cell solution in the thermocycler block.
Set up the thermocycler to heat the sample at 99°C for 10 minutes.
Mixing the Sample
After heating the sample for 10 min, we will prepare it again for centrifugation.
First, take the PCR tube out of the thermocycler block.
The block and the heated lid will still be hot, so take extra care.
Flick the PCR tube for 5 seconds to mix the sample.
Centrifuging the sample
In this step, you will spin the sample to separate the supernatant from the cell debris. Now the cells have burst thanks to the heating step, the DNA will be released from the cells and floating in the supernatant.
The molecular weight of DNA is lighter than the other cell material, like proteins and cell walls. By spinning the sample with centrifuge, we seperate the cell material from the DNA, which gives us a cleaner DNA sample.
To spin the PCR tube with your sample (3) in the Bento Lab’s microcentrifuge, you will need to use the PCR tube adapter (1) that sits in a normal microcentrifuge tube (2) and converts it to fit a PCR tube.
Remember to balance your centrifuge. So, if you are only working with one sample, prepare another PCR tube with an amount of water equivalent to your sample.
Set the centrifuge to run for 90 seconds at 8kG.
Cleaning up the sample for storage
After centrifugation, all the cell debris has been forced to the bottom of the PCR tube (1), leaving only the DNA in the liquid supernatant (2). The supernatant should look clear, like water.
Finally, you will transfer the supernatant into a new PCR using the micropipette.
Set the micropipette to 20μL and put on a new tip. Transfer 40μL of the clear supernatant into the new PCR tube.
Be careful to avoid pipetting any cell debris into the new tube. You should only transfer the clear liquid supernatant. Avoiding any of the cell debris will reduce the chance of interference with the DNA sample.
Labeling and storage
The new tube now contains only the DNA in the liquid. It is called the template sample, and can now be further used for analysis using protocols like PCR.
Label the tube again, so that you can identify which template sample it is.
Finally, if you are not using the template sample in another protocol right away, store it in the freezer at around -20°C. This will preserve the sample.
Although DNA itself is very stable, there might still be some other proteins in the sample that will degrade it over time. The purpose of this protocol is to clean up the saliva sample as much as possible whilst retaining the DNA. Storing the sample in the freezer will slow down any reactions from left over proteins and therefore the template DNA sample will be preserved longer.
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