human_DNA

Amplification of human genomic DNA

Last time we extracted our own genomic DNA. To determine whether the extracted DNA is of good quality, we tried to amplify a fragment of the human PDE1C gene by Polymerase Chain Reaction (PCR).

The two primers used were:

PDE1C_for: 5′-CTGGAACCAGTGCCACTATAA-3′; melting temperature of 54oC.

PDE1C_rev: 5′-CGACTTCAGCCATCTCTCTATC-3′; melting temperature of 55oC.

The conditions used for the PCR are accessible here.

We were expecting a 314 bp long fragment and the PCR products were run on an 1% agarose gel.

PCRgDNA_191114b

As expected, there was no band observed in the Negative Control since the kiwi does not contain the PDE1C gene which is always a good news. For our own genomic DNA, only the PC and M lanes produce the expected band. For the Y lane, we observed two bands at 100 bp which should correspond to some amplified DNA since the primers are only 21 bp long.

Since not all the extracted DNA worked as expected, we will try a new DNA extraction protocol.

Fingerers crossed we’ll get a hold of cheap method for extracting genomic DNA using kitchenware materials without the reliance on professional kits! This hopefully will give us the ability to test our own DNA for interesting genetic variants!

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Extraction of our (human) genomic DNA

After a preliminary successful extraction of DNA from kiwi with a standard protocol (here or here), we decided to try if we could do the same with our own DNA. The general principle is similar to extracting DNA from fruits except that we start with far less material.

Water_salt

The first step was to obtain some cells from our body! The easiest and quickest way is to retrieve cells present in our mouth. For this, we first prepare a mixture of water and salt (2 spoons of salt in 100 ml of tap water) and gargle it for 30-45 seconds.

The liquid now contains human cells and other stuff such as bacteria. The next step is to break the cells to release our genomic DNA in the solution. To do this, we just added a spoon of kitchen detergent to the solution and mix it.

AfterGargleDetergentAfterDetergent

SoySauce

To improve the visualisation of DNA, we also add some soy sauce to the solution to make it darker. However, since we retrieve at the end more DNA than expected, this step can be removed since it doesn’t help the final purification steps.

The penultimate step is to precipitate DNA by adding pure isopropanol to the solution and mix it. It generally takes a few minutes before the DNA start to became visible. We transferred some of our DNA to a clean tube with pipettes to keep it frozen until we test it by PCR with primers again a specific human gene to verify that we really have genomic human DNA (more about this next time).

IsoproHPIM2921HPIM2916

                                                           DNA is visible in the tip             DNA pellet with soy sauce

Just to compare to the amount of DNA retrieved from the kiwi extraction (all the white stuff in the bottle) and a photo of the material used for extraction:

                         HPIM2907     HPIM2926