DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

This video will demonstrate how to extract DNA from paraffin embedded tissue samples. The sample used in this demonstration is a tissue core, three millimeters in diameter, and six millimeters in length. While a single core is used in this demonstration, larger samples greater than five millimeters in diameter and 10 millimeters in length, or two to three cores of the size used here can be processed in a single reaction.

However, it is important to note that adjustments to the protocol may be required to ensure complete removal of paraffin and tissue digestion Before anything can be done to the tissue, the waxy paraffin preserving the tissue must be dissolved. This is accomplished with the use of xylene as xylene and phenol are toxic. Any work with these chemicals should be carried out in a fume hood, add 800 microliters of xylene to the tube containing your sample.

Then place the sample on a rocker for 10 minutes with gentle shaking. Spin down the sample at 14, 000 RPM for three minutes to pellet the sample and then remove xylene and dissolve paraffin from the tube. Repeat these steps two or three times until the paraffin is fully dissolved.

The tissue should appear wet and soft. It is important to remove all the paraffin to increase the final yield of DNA. Therefore, more washes are required for larger tissue Samples.

Now That the paraffin has been removed from the sample, the tissue must be rehydrated to allow for tissue digestion. This is accomplished using an ethanol rehydration series. First, add 800 microliters of 100%ethanol to the sample vortex and spin the sample at 14, 000 RPM for three minutes.

Remove the supernatant. Repeat these steps using 800 microliters of 70%ethanol, and then using 50%ethanol. The final spin should be five minutes long because the tissue will be less solid and requires more force to pellet.

Now the tissue needs To be digested to release the DNA from the cells. This is done using lysis buffer, a solution made of protein Dena, such as SDS and proteinase. K.The enzyme responsible for tissue degradation.

Add 200 to 500 microliters of lysis buffer to the sample. Depending on the size, 400 microliters is a good volume to use. For course, the components of lysis buffer as well as the incubation temperature of 56 degrees Celsius serve to maximize the activity of protein.

ACE K.Because protein, ACE K can only degrade tissue for so long, more enzyme must be added periodically to continue the reaction. 20 microliters of protein. ACE K at a concentration of 10 milligrams per M is added to the sample morning and evening until the tissue is completely dissolved.

When You can't see any More solid tissue, the DNA needs to be extracted from the cellular debris buffer. Saturated phenol is used to separate the sample into aqueous and organic phases. The aqueous phase contains the DNA.

It is important to use buffer saturated phenol because the acidity of normal phenol not only degrades your DNA, but also inverts the layering of the aqueous and organic phases. Be aware that buffer saturated phenol is stored in two phases. The phenol being the lower layer.

Add an equal volume of buffer saturated phenol to the sample and invert the tube several times. Next, place the sample in the centrifuge and spin it. 14, 000 RP RPM for five minutes to induce phase separation.

The DNA and RNA will be found in the upper aqueous phase, whereas protein, lipids and polysaccharides are found in the interphase and lower organic phase. Carefully remove the aqueous layer without disturbing the interphase and place it into a new tube. Often after the first spin, the interface is loose and back.

Extractions are required using small volumes of water. The protocol for back extractions can be found in the text accompanying this video. Repeat these steps until the interface is clear, which is usually achieved after three to five repetitions proceed by adding an equal volume of phenol chloroform, isoamyl alcohol to the DNA sample.

This serves the same purpose as phenol alone, but sharpens the interface to maximize the yield of DNA. Repeat this procedure once or twice until the interface is clear and sharp. Now that your sample is free of protein, any contaminating RNA has to be removed.

This is achieved by RNAs. A degradation RNAs A is added to the sample at a final concentration of 100 micrograms per mil, and the sample is incubated for one hour at 37 degrees Celsius. Now that the RNA is degraded, the RNAA enzyme has to be removed.

The DNA cleanup procedure is repeated as before. Using buffer saturated phenol first and then proceeding to phenol chloroform isoamyl alcohol, fewer cleanup steps will be required since the only contamination is now the RNA A enzyme and the degraded RNA. Now that the sample contains Only DNA, the DNA must be precipitated from solution add one 10th, the volume of sodium acetate and one volume of isopropanol.

Alternatively, two and a half volumes of ethanol can be added, but a larger tube would then be required. Incubate the sample at minus 20 degrees Celsius for 30 minutes to an hour, or if you are working late overnight. Now that you are working with DNA, make sure the sample remains on ice so that it doesn't degrade.

Spin down the sample at 14, 000 RPM for 10 minutes at four degrees Celsius to pellet the precipitated DNA. Remove the supernatant taking care not to dislodge the pellet to remove excess salts that could interfere with later experiments like sequencing. For example, add one mil of chilled, 70%ethanol and gently resus, suspend the sample.

Spin down the sample once again at 14, 000 RPM for 10 minutes at four degrees Celsius. Carefully remove the supernatant and let any excess ethanol air dry for up to five minutes. It is important not to over dry the sample.

Finally, the DNA is purified and is ready to be resuspended in your buffer of choice. Here we are using autoclave distilled water. Oh yeah.

Store DNA samples at minus 20 degrees Celsius for later use. After extraction is complete, you must assess the quality and quantity of your DNA. This is done by spectrometry you using the NanoDrop three 30 nd set the measurement type to nucleic acid, select DNA 50 as a calculation and initialize the instrument with 1.5 microliters of water.

Clean the pedestal and take a blank measurement using 1.5 microliters of diluent. In this case distilled water. Clean the pedestal and load 1.5 microliters of sample and hit measure.Yep.Great.

An A two 60 over two 80 ratio of 1.8 is ideal at this point. Your DNA is ready for use in downstream applications, which include but are not limited to. Array, comparative genomic hybridization, methylated, DNA, immunoprecipitation and sequencing.

The links for these protocols are shown here, if not needed immediately. Store your samples at minus 20 degrees Celsius for later use.