Rhesus Blood Group

Protocol

In this project, you will first extract human DNA from saliva. This will take about 20 minutes. After this, you will use PCR to amplify the D gene and the CcEe gene. This will take about 120 min, but most of it will be waiting time. Finally, you will visualise the results using Gel Electrophoresis, which will take about 45 min. At the end of each section, you can continue right away, or store your samples and continue later.

DNA Extraction

First, obtain the DNA sample. Use the DNA Extraction from saliva protocol. It will take ca 20 min, at the end of which you should have a clean DNA template sample in a PCR tube.

PCR

In this step, you will use PCR to amplify both the D and CcEe gene of the Rh gene cluster. It is possible to use the same primers to amplify both the D gene and CcEe gene, as the DNA sequences are very similar (homologous).

Because the CcEe gene is comparatively long, the duration of the annealing and extension steps have been set to 60 and 90 seconds, instead of the usual 30 seconds.



You will need the DNA template sample (1), an empty PCR tube (2), the primer mix for this project (3), the mastermix (4), and PCR grade water (5). The total final volume of your tube will be 20μL.

First add the mastermix. Set your micropipette to 4μL.

Using a fresh pipette tip, transfer 4μL of the master mix into the empty PCR tube. Then discard your tip.

Next add the primer mix. Set your micropipette to 2μL. Using a fresh pipette tip, transfer 2μL of the primer mix into the PCR tube. Then discard your tip.


Now add the DNA template. Set your micropipette to 4μl.

Using a fresh pipette tip, transfer 4μl of the DNA template sample from the sample tube into the PCR tube with the mastermix and primer mix. Then discard your tip.

Finally add PCR grade water to make the total volume up to 20μl. Set your micropipette to 10μl.

Using a fresh pipette tip, transfer 10μl of PCR grade water into the PCR tube. Then discard your tip.

Place your PCR tube in the thermocycler block.

Set up the thermocycler with the following PCR program:


• 120 sec at 94°C
• 35 cycles made of 3 steps
  • 30 sec at 94°C
  • 60 sec at 64°C
  • 90 sec at 72°C
• 5 min at 72°C



If you need help operating the Bento Lab thermocycler, check the manual. You can use the PCR  preset (1), then modify (2) the program to the required settings (3) before running the program (4).


The program will run for ca 2 hours. When it is finished, you can keep the result in the freezer, or use it right away for gel electrophoresis.

Gel Electrophoresis

Follow the Gel Electrophoresis Protocol to cast a gel and run it with your PCR result, and a 100bp ladder. This should take about 40 min.

Visualising the Gel

After the gel run has completed, you can visualise your results.

Continue to wear gloves as you handle the gel.

Open the orange lid of the gel box, and wipe off the condensation.  


Gently pour out the buffer, and dispose of the buffer down a drain.

Drain disposal of TBE running buffers is a standard waste disposal procedure followed by research labs. If you have questions, get in touch with us.

Place the gel box onto the Bento Lab transilluminator surface. In order to get best visibility, you should do this in a room as dark as possible.


Turn Bento Lab on, select the Gel Electrophoresis module, and turn on the Transilluminator light.


Hold the orange filter lid over the gel to visualise the DNA bands. For documentation, use your mobile phone to take a clear picture of the gel. Rather than holding the lid over the gel, you can hold the lid directly in front of your camera lense.

If the bands are faint, try to reduce the light in the room, e.g. by closing the curtains and turning off the lights.  You can also carefully take the gel out of the gel box and place it directly onto the transilluminator. Wear gloves when doing this, and be careful not to break the gel.

Analysing your results

Compare the picture of your gel to this example result, which has been run with all variations. Your sample should correspond to one of these variations.



1 – Ladder – 100 bp DNA Ladder

2 – Result Rh-
CcEe gene (1200 bp)
This result shows a person who has no copies of the D gene, so homozygous recessive. This person will have an Rh- blood type.

3 – Result Rh+
CcEe gene (1200 bp), D gene (600 bp)
This result shows a person with one or two copies of D gene, so someone who is heterozygous or homozygous dominant. This person will have an Rh+ blood type.

Disposal of agarose gels is a standard waste disposal procedure followed by research labs. If you have questions, get in touch with us.