Hi, I'm Jody from the laboratory, Steven Hallam in the Department of Microbiology in immunology at the University of British Columbia. Today we'll show you a procedure for extracting genomic DNA, concentrated from seawater samples onto 0.2 micron STX filters and a purification of this DNA by a cesium chloride density gradient centrifugation. We use this procedure in our laboratory to obtain environmental DNA so that we can study microbial community structure, diversity and metabolism in marine environments.
So let's get started. Begin this protocol by thawing on ice, the stir of X filters that you have previously concentrated with planktonic biomass, treated with lysis, buffer and frozen for simplicity. Here we describe the procedure for processing an individual filter.
In practice we recommend processing 15 or fewer filters at a time. Once the filter has thawed, begin the first of two incubations. One to lyce the cells and remove RNA and one to break down proteins.
For the first incubation, add 100 microliters lysozyme, and 20 microliters RN a's a to the filter. Reseal the filter with parfum and leave it to rotate at 37 degrees Celsius for one hour. For the next incubation, add 100 microliters protein ace KN 100 microliters 20%SDS to the filter.
Reseal the filter using perfil and leave it to rotate one to two hours more. This time at 55 degrees. Use a five cc syringe to transfer the lysate from the Stax filter to a 15 milliliter falcon tube.
Rinse the filter with one milliliter lysis buffer and add the rinse liquid to the lysate in your falcon tube. Now that you have lysed and digested your cells, you are ready to extract their DNA. The next step is to use the phenol chloroform method to extract the DNA from your lysate.
Add an equal volume about three milliliters of phenol chloroform, isoamyl alcohol to the lysate tube vortex. For 10 seconds to mix well spin the tube at 2, 500 times G for five minutes. During centrifugation, the phenol chloroform lysate mixture should separate into two layers.
An organic layer on the bottom containing the phenol chloroform and proteins from your sample. And an aqueous layer usually on top, which consists of your DNA water and other more hydrophilic molecules. Transfer the aqueous layer containing your DNA into a new 15 milliliter Falcon tube To this tube, add an equal volume of a chloroform isoamyl alcohol mixture vortex for 10 seconds.
This step removes any remaining phenol from your DNA sample. Spin it 2, 500 times G for five minutes or until the aqueous layer is clear. Transfer the aqueous layer into a new labeled falcon tube and add one milliliters of TE at PH eight.
Now you have your DNA extract. The next step is to wash and concentrate the DNA sample in a 15 milliliter Amazon ultracentrifuge tube. Transfer your DNA sample to the filter compartment of anon.
Ultra tube theon tubes consists of a filter that retains molecules at or above a specified molecular weight, the tate and a tube to catch the flow through the filtrate. In this case, the filter retains your DNA spin at 3, 500 times G for 10 minutes. Check to make sure there is less than one milliliter of liquid retained in the Amon filter.
If more reten Tate remains, refill the filter with TE and spin again. Remove the flow through or filtrate to another falcon tube and save it in the fridge until you have confirmed your final product on a gel, add two milliliters TE buffer to the Amazon filter and spin at 3, 500 times G for six minutes. Remove the filtrate as before.
Wash the DNA twice more with TE for a total of three washes, saving the filtrate each time for the last wash spin until 200 to 500 microliters of Tate remain in the Amon filter. Note the final volume and transfer to a labeled 1.5 milliliter einor tube. Make a 70 microliter aliquot of the washed concentrated DNA to use as working stock.
Store the working stock at minus 20 degrees Celsius. Freeze away the rest of the DNA at minus 80 degrees Celsius. Check the DNA concentration and quality of your final product by running out your samples on a gel next to DNA.
Ladders which serve as size and intensity standards set up a 0.8%AROS gel plus atherium bromide to run overnight. In the first few lanes load ladders with bands of various molecular weights and concentrations, we recommend the following loading pattern. In the first two lanes, load 10 microliters of one kilobyte plus ladder in the next three lanes, load two microliters, five microliters, and 10 microliters respectively of 50 nanograms per microliter Lambda H three ladder.
Lastly, load five microliters per lane of DNA extract sample. Run the gel at 15 volts for approximately 16 hours. Photograph the gel using the UV gel documentation system to determine the molecular weight range of your DNA and its concentration.
Compare the sample bands to bands of the ladder. Good quality DNA will have high molecular weight and show little evidence of shearing or degradation. If the DNA quality is good and quantity is sufficient, proceed to purify the DNA by performing a cesium chloride gradient.
Centrifugation first label, one centrifuge tube for each sample to each centrifuge tube, add 160 milligrams of cesium chloride and 178 microliters of genomic DNA. Cover the tube with a small piece of perfil and gently invert the tube 10 to 20 times in order to mix the components. Do not mix by pipetting which may cause shearing of the DNA.
Then add 10 microliters of AUM bromide to the tube and mix again. Make sure that the tubes are balanced well before centrifugation and that the weight differences among the tubes are less than one milligram. Put the tubes into the ultracentrifuge rotor using tweezer scissors.
Then close the rotor lid and place the rotor inside the ultracentrifuge close. The ultracentrifuge door vacuum will be applied as soon as you close the door. Run overnight for a total of 18 hours at 100, 000 RPM at 20 degrees Celsius.
When centrifugation is complete, take out the tubes from the rotor using tweezer scissors and place on the rack. Take the tubes over to the blue light transluminator, put on the amber filter glasses. Then turn on the blue light and behold your DNA band using a sterile one cc syringe n needle.
Remove the DNA band and then place it in a 1.5 milliliter einor tube to help recover most of the DNA trapped in the dead space in the syringe. Rinse the syringe with 100 microliters te and add the rinse solution to the tube. Prepare one tube with 100 microliters te buffer per sample in order to prevent cross-contamination.
To remove AUM bromide from the DNA, add an equal volume of water saturated butanol to the tube. Invert the tubes gently, 10 to 20 times centrifuge at 10, 000 RPM for one minute and discard the top layer. Repeat washing about three to four more times until the color of the butanol is transparent.
Place four milliliters of TE in ancon ultracentrifuge tube and add the DNA solution from above centrifuge at 3, 500 times G at room temperature until the DNA volume is reduced to approximately 100 to 500 microliters and discard the flow through. Add two milliliters TE to the Amazon filter and centrifuge at 3, 500 times G for six minutes. Repeat twice for a total of three washes with te concentrate to a final volume of 50 to 100 microliters by additional centrifugation as necessary.
And transfer the DNA solution on the filter to a new 1.5 milliliter einor tube. Add 40 microliters te to theon filter and pipette up and down along both filter membranes to wash out any remaining DNA. Add this solution to the tube of concentrated DNA.
Place a MicroCon YM 30 filter unit to a MicroCon tube and pre-wash the MicroCon by adding 200 microliters autoclave water and centrifuging at 10, 000 RPM for seven minutes. Add the DNA solution to the pre-washed MicroCon in order to further concentrate the DNA centrifuge at 5, 000 to 10, 000 times G for one to three minutes. Check the amount of liquid on the filter and repeat centrifugation until the amount of liquid on the filter is reduced to approximately 50 microliters.
Place the filter unit upside down in a new MicroCon tube and centrifuge at 1000 times G for three minutes. The ideal amount of concentrated solution is 50 to 60 microliters. Transfer your DNA sample to a new 1.5 milliliter einor tube.
Measure the concentration of DNA on a NanoDrop and check the peak quality. A graph of absorbances for clean DNA should have a clear peak at 260 nanometers. Freeze the rest of the DNA at negative 80 degrees Celsius.
When this protocol is done correctly, you should see a gel image similar to figure one actual DNA concentration of extracts will vary depending on the source of the sample. When this protocol is done correctly, you should clearly see a DNA band across the middle of the tube. As in figure two, We've just shown you how to extract genomic DNA from sterex filters and purify it using a caesium chloride density gradient.
When doing this procedure, it's important to check before you begin that you have all the necessary reagents and expendable items. And secondly, plan at least three full days to allow plenty of time to complete the procedure carefully. So that's it.
Thanks for watching and good luck with your experiments.
