Genomic DNA Extraction Protocol (High molecular weight DNA for sequencing)

##### Updated by E. Hiebert and J. Kavaler, Summer 2023. **Fundamentals:** Genomic DNA extraction from cells involves four basic steps - 1) isolation and homogenization of tissue/cells 2) Lysis of cells 3) Phenol/chloroform extraction of proteins/other cell components 4) Ethanol precipitation of purified DNA. :::info - It is important to keep samples and tubes cold (on ice) as much as possible - Wear protective equipment (gloves, lab coat, safety glasses) when using phenol. Phenol is strongly caustic and will burn your skin if you come in contact with it. PROTECT YOUR EYES. It will eat through your clothing. Chloroform is not caustic, but is a probable carcinogen and vaporizes easily. Use in a fume hood. Be careful with both chemicals. Dispose into a hazardous waste container. ::: **I. Isolation of samples** The source of your DNA can determine the ease of DNA extraction. Sperm is an ideal source as it consists of individual cells that consist of primarily a nucleus and so are already somewhat enriched for DNA. DNA can also be extracted from tissue but the tissue has to be broken down to generate a single cell (or nuclear) suspension. :::info If you wish to measure the weight of your sample, pre-weigh the final tube that will hold the sperm or tissue and weigh it again after collection. ::: *A. For Ciona sperm:* 1. Set up 60 mm petri dish with 1 ml of filtered sea water (FSW). 2. Take animal and remove tunic. 3. Collect eggs as usual (these can be used for fertilizations or simply discarded). 4. Collect sperm into petri dish. 5. Move sperm from petri dish into 2 ml microfuge tube. 6. Centrifuge to pellet sperm at 3000xg for 3 minutes at 4°C and discard supernatant. 7. Resuspend sperm in 1 ml of chilled FSW. Make sure they are resuspended well and not clumpy (Vortex if you have to). 8. Sediment eggs and large contaminating tissue by allowing the tubes to stand for 4 minutes on ice. 9. Carefully transfer the sperm suspension to a fresh microfuge tube, avoiding the sedimented material. 10. Repeat steps 6 through 9. 11. Do a final spin at 3000xg for 3 minutes at 4°C and remove as much of the supernatant as possible. 12. Proceed to cell lysis OR for storage, flash-freeze the sample in LN~2~ and store at -80°C until use. *A. For Ciona tissue:* 1. Take animal and remove tunic. 2. Using dissection scissors, cut off tissue from the animal, preferably muscle/body wall/siphons. Avoid guts. (This step can be done after sperm and/or eggs have been collected). Weigh tissue now if desired. 3. Place tissue on a chilled, flat, clean surface (I use a glass plate, dissecting dish, or microscope slide) and mince with scissors as much as possible. (Alternatively, one can place tissue in a mortar with LN~2~ and grind with a pestle) 4. Move tissue from the surface to a pre-chilled Dounce homogenizer tube on ice [(FisherSci)](https://www.fishersci.com/shop/products/kimble-chase-kontes-dounce-tissue-grinders-7/K8853000007). Add 5 ml of pre-chilled Homogenization Buffer (see below). Use the fine pestle (will disrupt tissue and cells but keep nuclei intact) and slowly move the pestle up and down. This will be difficult and you will have to fight the tight seal of the homogenizer. Move slowly or it will splash when suction is overcome. If you have a lot of tissue and/or movement is very difficult, either mince your sample better or start with the coarse pestle. Keep on ice as much as possible while you homogenize. 5. Place homogenate in a pre-chilled 15 ml conical tube and spin at 1000 RPM for 1 minute at 4°C to pellet debris. 6. Move the supernatant to a fresh pre-chilled 15 ml conical tube. Spin at 4500 RPM for 10 minutes at 4°C. The pellet from this spin should contain nuclei and individual cells from your homogenate. 7. Discard supernatant, resuspend sample in 500 µl of Homogenization Buffer, transfer to a 1.5 ml microfuge tube and proceed to cell lysis. If you prefer to store the sample, pellet the nuclei cells again, flash-freeze the sample in LN~2~ and store at -80°C until use. **II. Lysis of samples** :::info Homogenization Buffer (20 ml):       0.2 ml        1M Tris pH 7.5       0.24 ml      5M NaCl       0.4 ml        0.5M EDTA pH 8.0       83.3 µl       0.3M spermine       25 µl          1M spermidine ::: 1. Resuspend samples in 0.5 ml Homogenization Buffer (thaw first if working with frozen stocks). If working with fresh tissue, you will have already done this step. 2. Add 5 µl of 20 mg/ml proteinase K and 50 µl of 10% SDS and mix by gently inversion. The solution will likely get somewhat viscous now, depending on the amount of starting tissue. Incubate at 37°C for 1 hour, gently mixing tube on occasion. 3. Add 0.5 ml of 1:1 phenol/chloroform. Gently mix (it should become cloudy). Chill tubes on ice for 2 minutes. Spin at high speed (16,000xg) for 2 minutes. The phases should separate - the bottom phase will be phenol/chloroform and the top layer will be the aqueous layer. Carefully remove the aqueous phase to a fresh 1.5 ml microfuge tube. 4. Repeat Step 3. 5. Add 0.5 ml of chloroform. Gently mix (it will become cloudy but the phases will begin to separate almost immediately). Chill tubes on ice for 2 minutes and spin at high speed for two minutes. Carefully remove the aqueous (top) phase to a fresh 1.5 ml microfuge tube. 6. Add NaCl to a final concentration of 200 mM. Usually there is some volume loss during the extractions. Assuming you retain 400 µl of DNA solution, add 16 µl of 5M NaCl. Then add two volumes of 100% ethanol (800 µl for a 400 µl solution. 7. Mix gently. Ideally, you can see the strands of DNA precipitate. If large enough, these can be removed via “fishhook” – using a sealed, bent end of a pasteur pipet – washed in 70% ethanol, dried and placed in your choice of H2O, Elution Buffer, TE, etc. 8. If you can’t/don’t want to use a fishhook, spin the tube at 16,000xg for 10 minutes. Remove supernatant – you should see a pellet. This method will likely bring down any contaminating RNA but is necessary for small yields. RNA can later be removed by incubation with RNAse A if necessary. 9. Wash pellet with 1 ml of 70% ethanol and repeat high speed spin (5 minutes). Remove as much supernatant as possible and air dry the pellet. 10. Dissolve the dried pellet in either H2O, Elution Buffer, or TE (depending upon concentrantion needs, anywhere from 50 to 400 µl). It may take some time for the DNA to dissolve, which can be done overnight in the refrigerator if you’re not in a hurry. 11. Take a nanodrop reading on the DNA to determine concentration.