and connect with us on
This protocol describes how to extract a DNA sample from cheek cells in human saliva using the HotSHOT DNA Extraction Kit and Bento Lab.
It is an alternative method to the DNA Extraction from Saliva.
You will use 1.5 mL tubes to extract the DNA samples from cheek cells 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.
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 (approximately 0.3 g) with approximately 33 mL tap or bottled water. A large shot glass (35 mL or 50 mL using a large pinch or pinch and a half of salt respectively) is perfect for this. This will produce an approximately 0.9% saline solution.
For better accuracy you could weigh both the salt and water in larger amounts with kitchen scales, for example adding 4.5 g of salt to a 500 mL bottle of water.
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 create an osmotically balanced solution to help protect cells prior to DNA extraction, reducing the chance that the DNA will be degraded before the heat extraction step.
Measure approximately 10 mL salt water into a shot glass, and pour this into your mouth. Rinse your inner cheeks vigorously for 60 seconds, swishing the water from side to side and rubbing your cheeks against your teeth to dislodge cheek cells.
When you are done, spit the saline solution back into the glass.
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.
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 centrifugal 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. If you are using Bento Lab Pro, set the speed to 4,000 ×G and spin for 90 seconds. If you’re using Bento Lab Entry, spin for 5 min (at 2.7k ×G).
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 liquid (the supernatant) away. This should leave a very small amount of liquid. Try to remove as much liquid as possible without dislodging the pellet.
Now carefully pipette all of the remaining liquid off without disturbing the pellet, and discard the liquid.
You should now have a white pellet in your sample tube. It should be about the size of a matchstick head (around 2-3 mm diameter), visible when held to the light or against a black background, and flattened against the inside tip of the tube.
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.
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, and then centrifuge again.
Once you have a large enough pellet and have removed as much of the supernatant as you can, add 80 µL of the HotSHOT Alkaline Lysis Solution.
In this next step, you will use the micropipette (1) to transfer 80 μl. of the HotSHOT Alkaline Lysis Solution to the 1.5 mL tube containing the cell pellet (2). You will then resuspend the pellet in the buffer and transfer it into a 0.2 mL PCR tube (3), so that you can heat it in the thermocycler.
First, if you only have a 20 μl pipette, set the adjustable pipette to the maximum volume of 20 μl.
Make sure the pipette has a new pipette tip. Then use the pipette to transfer 20 µL of HotSHOT Alkaline Lysis Solution into the mouth of the 1.5 mL sample tube by expelling the droplet directly over the mouth of the tube without touching the sides.
Repeat this step four times to achieve a final volume of 80 μL, using the same tip. If the tip does touch the sides of the tube, change the tip to avoid contamination of the HotSHOT Alkaline Lysis Solution.
If you have a 200 μl adjustable pipette, you can set it to 80 μL as shown above, and pipette the whole volume all in one go.
Never put a pipette tip that has touched any surface, liquid, or solid, into a bottle or tube, unless you intend to transfer that substance back into that container. If you do, you run the risk of contaminating the bottle or tube.
To minimise the chance of contamination of the HotSHOT kit, you could carefully pour or pipette the volume needed for all samples into another tube in advance.
You now have a concentrated cell sample in 80 µL of HotSHOT Alkaline Lysis Solution.
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 HotSHOT Alkaline Lysis Solution.
If you are working with more than one sample, you can repeat the addition of HotSHOT Alkaline Lysis Solution for each sample using the same pipette tip, provided you don’t touch the sides of the 1.5 mL sample tubes. If you do touch the sample tubes, change the tip to prevent possible contamination of the HotSHOT Alkaline Lysis Solution.
You can leave the pipette tip on the micropipette for the next step to minimise plastic waste.
First, if you have only a 20 μL pipette, set the adjustable pipette to the maximum volume of 20 μl, and use the same pipette as you used in the previous step.
Carefully, pipette up the contents of the 1.5 mL sample tube and transfer it to a 0.2 mL PCR tube in 20 μl increments until all the liquid has been transferred.
If you have a 200 μL pipette, you can set the pipette to 80 uL and pipette this volume all in one go.
You can now dispose of the pipette tip. If you are working with more than one sample, you must use a new tip to transfer each sample into the 0.2 mL PCR tube to avoid cross-contamination.
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.
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 98° C for 10 minutes.
After heating the sample for 10 min, we will neutralise the sample extraction and 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.
The heat may have caused some tubes to loosen, so press down on the lids before removing them.
Once you have removed the tubes, allow them to cool.
Then using the micropipette as before, and a new pipette tip, add 80 μl of HotSHOT Neutralising Buffer to each tube by pipetting into the open mouth of the PCR tube, avoiding touching the sides of the tubes. If you do touch the sides of the tubes or anything else, replace the pipette tip.
Never put a pipette tip that has touched any surface, liquid, or solid, into a bottle or tube, unless you intend to transfer that substance back into that container. If you do, you run the risk of contaminating the bottle or tube.
Then close the lid firmly, and flick the PCR tube for 5 seconds to mix 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 and alkaline lysis 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 the centrifuge we separate 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.
If using Bento Lab Pro, set the centrifuge to run for 90 seconds at 8k ×G. If you’re using Bento Lab Entry, spin for 5 min (at 2.7k ×G).
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 tube using the micropipette.
If you only have a 20 μL micropipette, set it to 20μL and attach a new tip. Transfer 40 μL of the clear supernatant into the new PCR tube by pipetting twice.
If you have a 200 μL micropipette, you could also set it to 40 μL and transfer the whole 40 μL of supernatant in one go.
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.
The new tube now contains primarily only the DNA stored in the neutralised HotSHOT solutions. The HotSHOT Neutralisation Buffer present in the solution acts as a pH buffer, allowing it to be stored for longer without degradation.
The DNA solution is called the template sample, and it can now be further used for DNA 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 help preserve the sample.
Although DNA itself is very stable, heating has transformed it from a double-stranded form to a less stable single-stranded form, and it is still vulnerable to degradation by enzymes and other processes.
Fortunately, the neutralised HotSHOT solutions contain a pH buffer to reduce DNA damage, and a chelator (ethylenediaminetetraacetic acid or EDTA) that binds to and removes magnesium ions from solution and thereby prevents any DNA-degrading enzymes from functioning.
Storing the sample in the freezer will slow down any reactions from left-over enzymes even further, and therefore the template DNA sample will be preserved longer.
