Extraction of DNA basically consists of four major steps.
Preparation of a cell extract
Purification of DNA from cell extract
Concentration of DNA samples
Measurement of purity and DNA concentration
To extract DNA from a tissue/cells of interest, in this experiment the heart muscle of rats, the cells have to be separated and the cell membranes have to be disrupted. The "Extraction buffer" helps in carrying out these processes. Chemicals such as EDTA (Ethylene Diamine Tetra Acetate) which removes Mg2+ ions that are essential for preserving the overall structure of the cell membrane, and SDS (Sodium Dodecyl Sulfate) which aids in disrupting the cell membranes by removing the lipids of the cell membranes are included in the extraction buffer. Having lysed the cells, the final step in the preparation of a cell extract is removal of insoluble cell debris. Cell debris and partially digested organelles etc. can be pelleted by centrifugation leaving the cell extract as a reasonably clear supernatant.
In addition to DNA the cell extract will contain significant quantities of protein and RNA. A variety of procedures can be used to remove these contaminants, leaving the DNA in a pure form. The standard way to de-proteinize a cell extract is to add phenol or a 1:1 mixture of phenol:chloroform. These organic solvents precipitate proteins but leave the nucleic acids in aqueous solutions. The aqueous solution of nucleic acid can be removed with a pipette. The effective way to remove RNA is with the enzyme ribonuclease, which will rapidly degrade these molecules into ribonucleotide subunits.
The most frequently used method of concentration is ethanol precipitation. In the presence of salt and at a temperature of -20 °C or less, absolute ethanol will efficiently precipitate polymeric nucleic acids. With a concentrated solution of DNA one can use a glass rod to pull out the adhering DNA strands while for dilute solutions precipitated DNA can be collected by centrifugation and redissolving in an appropriate volume of water.
DNA concentrations can be accurately measured by UV absorbance spectrometry. The amount of UV radiation absorbed by a solution of DNA is directly proportional to the amount of DNA sample. Usually absorbance is measured at 260 nm, at which wave length an absorbance of 1.0 corresponds to 50 µg of double-stranded DNA per ml. UV absorbance can also be used to check the purity of a DNA preparation. With a pure sample of DNA the ratio of the absorbancies at 260 nm and 280 nm (A260/A280) is 1.8. Ratios of less than 1.8 indicate that the preparation is contaminated, either with protein or with phenol.
The isolation of genomic DNA from rat heart muscle will be carried out using the Sigma GenElute Mammalian Genomic DNA Miniprep Kit and an adapted protocol. This kit uses a convenient spin-column format to isolate the genomic DNA as opposed to the more traditional methods outlined in your notes.
Once tissue is disrupted by grinding in liquid nitrogen, chemical digestion is initiated by the addition of a tissue lysis solution and Proteinase K. The addition of a cell lysis solution containing chaotropic salts further denatures macromolecules and completes the process to free the genomic DNA. The addition of ethanol before centrifugation causes the DNA to bind to the silica matrix found in the binding column. Washes are carried out to remove contaminants and the DNA is eluted through the column in a high pH buffer.
YOU MUST COMPLETE STEPS 1-3 DURING THE MORNING TUTORIAL HOUR!
Please Dispose of All Used Materials and Media as Biohazard Waste
1. In a sterile 1.5 ml Eppendorf tube, add 180 µl of Lysis Solution T (for tissue) and 20 µl of Proteinase K solution. Keep on ice.
2. Obtain about 50 mg of frozen rat heart muscle, you will lose upto 50% during the homogenization and transfer process. Place in a mortar and flash-freeze with liquid nitrogen. Do not let the liquid nitrogen completely evaporate until homogenization is complete. Use your judgment, too much liquid nitrogen in the mortar will make it hard to forcefully grind the tissue as it will splatter when you try to crush larger bits of muscle. Grind tissue with mortar and pestle until it is a fine powder.
3. Use a small spatula and quickly scrape the frozen, powdered tissue into the pre-prepared tube containing Lysis Solution T/Proteinase K. Incubate at 55 °C until the tissue is completely digested - at least 2 hours (up to 4 hours). Vortex occasionally.
During this 2 hour incubation, you may proceed with the plasmid isolation portion of the lab. Otherwise, continue this procedure during the afternoon lab period and then begin the plasmid isolation protocol.
4. After the incubation period, cool the solution to room temperature. Add 20 µl of RNase A and let sit for another 2 minutes at room temperature.
5. Add 200 µl of Lysis Solution C (to lyse actual cells) to the sample and vortex for 15 seconds. Place in a 70°C heat block for 10 minutes and proceed with Step 6.
6. Add 500 µl of Column Preparation Solution to a pre-assembled GenElute Miniprep Binding Column (with a red O-ring). Centrifuge at maximum speed (13000 RPM) for 1 minute and discard flow-through.
7. Add 200 µl of ethanol to the lysate and vortex for 5-10 seconds until the solution is homogenous. Alternatively, mix by taking up and releasing with a pipette.
8. Transfer the lysate to the binding column and centrifuge for 1 minute.
9. Discard the collection tube containing flow-through liquid and place the column in a new 2 ml collection tube. Add 500 µl of Wash Solution to the binding column and centrifuge again for 1 minute. Again, discard the collection tube containing the flow-through liquid and again place the column in a new 2 ml collection tube.
10. Repeat the wash step using 500 µl of Wash Solution and spin at maximum speed for 3 minutes. Discard only the flow-through (not the collection tube) and spin the binding column in the empty collection tube for an additional 1 minute to make sure there is no residual ethanol from the Wash Solution. Finally, place the binding column in a new 2 ml collection tube.
11. Pipette 100 µl of Elution Solution (Tris-EDTA buffer, pH 9.0) directly into the centre of the binding column and let sit at room temperature for about 5 minutes. Centrifuge for 1 minute. Discard the column - the eluate contains the DNA.
12. Measure the absorbance of your genomic DNA sample at 260 and 280 nm and calculate the concentration. Be sure to use the same Elution Solution as a blank for the spectrophotometer in the same dilution ratio as your sample.
Please Dispose of All Used Materials and Media as Biohazard Waste
1. Pellet the E. coli cells from a 5 ml overnight culture. Take a 1.5 ml aliquot of the cell culture in a 1.5 ml Eppendorf tube and centrifuge at maximum speed for 1 minute. Discard the supernatant (culture medium) and repeat with the remainder of the culture. Each time you add culture medium to the tube, try mix the pelleted cells back into solution before spinning.
2. Resuspend the pelleted bacterial cells in 250 µl of Buffer P1. Ensure that RNAse has been added to buffer P1. No cell clumps should be visible.
3. Add 250 µl of Buffer P2 to lyse the cells. Mix gently by inverting the capped tube six times. Do not vortex. Incubate at room temperature for less than 5 minute.
4. Add 350 µl of Buffer N3 to neutralize the lysis reagent and mix immediately by inverting the tube six times. Do not vortex. Centrifuge the mixture at maximum speed for 10 min.
5. Place a spin cartridge in a 2 ml collection tube. Load the supernatant from Step 4 into the spin cartridge. Centrifuge at maximum speed for 1 minute. Discard the flow-through (not the collection tube!).
6. Place the spin cartridge back into the 2 ml collection tube and add 500 µl of wash Buffer PB to the spin cartridge. Centrifuge at maximum speed for 1 minute. Discard the flow-through.
7. Wash again by adding 750 µl buffer PE to the cartridge. Spin for 1 minute and discard the flow-through. Spin again for 1 min to remove residual buffer.
8. Place the spin cartridge into a new 1.5 ml Eppendorf tube. Add 75 µl of EB buffer directly to the center of the spin cartridge. Incubate at room temperature for 1 minute then centrifuge at maximum speed for 1 minute. The flow-through contains the eluted plasmid.
9. Check purity and concentration of the DNA by measuring the absorbance at 260 and 280 nm.
1. Turn on the spectrophotometer and the UV lamp. Set the wave length at 260 nm. Let warm up for at least 5 min.
2. Prepare blank with 20 µl of EB Buffer mixed with 980 µl of sterile water.
3. Dilute your sample in the same ratio: 20 µl of EB Buffer mixed with 980 µl of sterile water.
4. Using UV-permeable cuvettes, zero the spectrophotometer with the blank solution. Measure the absorbance of the diluted DNA preparation.
5. Change the wavelength to 280 nm.
6. Re-zero with the blank.
7. Take the absorbance of the diluted DNA preparation at 280 nm.
8. Calculate the purity and concentration of the DNA in your preparation.
Hint: 1 µg/ml = 0.001 µg/µl = 1 ng/µl. Using units per µl will make it easier to calculate the amount of DNA to add in subsequent reactions.
If your DNA concentrations are too low, we will re-measure the concentrations using the Nanodrop spectrophotometer. The Nanodrop has a lower detection threshold and is able to obtain accurate readings using only 1-2 µl of sample.
Properly label the tubes containing your genomic DNA and plasmid DNA. Store them in the fridge at 4 °C.