Lab 8: DNA Isolation
Lab 8: DNA Isolation
Introduction
The purpose of this lab was ultimately to familiarize ourselves with the PowerSoil DNA Isolation Kit to successfully purify a DNA sample from a pure E. coli culture in LB broth.. The first step will be obtaining the cells, then through use of the kit and instructions we will break down the membrane and release the DNA from the cells. The next step will be to precipitate the DNA and finally, purify the sample. "The PowerSoil DNA is intended for use with environmental samples containing a high humid acid content including difficult soil types such as compost, sediment and manure...The isolated DNA has a high level of purity allowing for more successful PCR amplification of organisms from the sample." (MoBio) Since the sample we are using is already a pure E. coli sample, all of the purification is a tad overkill but will be done anyways according to the provided instructions. Another important thing to note is that we do not know exactly what is in the various solutions we will be using because it is proprietary knowledge, owned by MoBio Laboratories.
From MoBio Laboratories: "The PowerSoil® DNA Isolation Kit is effective at removing PCR inhibitors from even the most difficult soil types. Environmental samples are added to a bead beating tube for rapid and thorough homogenization. Cell lysis occurs by mechanical and chemical methods. Total genomic DNA is captured on a silica membrane in a spin column format. DNA is then washed and eluted from the membrane. DNA is then ready for PCR analysis and other downstream applications."owerSoil
Methods and Materials
0.25 mL of the E. coli/LB broth was added to each of the two provided PowerBead Tubes that included a little bit of buffer solution and small crystals. The tubes were gently vortexed.
The buffer will help disperse the particles, begin to dissolve the humic acids (if this was a soil sample) and protect the nucleic acids from degradation. The vortexing gently mixes the solution and begins to break down the cell walls.
60 μL of Solution C1 is added to each tube and gently vortexed for 30 seconds. The tubes were then placed in a centrifuge and centrifuged at 10,000 x g for 30 seconds at room temperature.
This initial centrifuging was the first step to precipitate out the non DNA organic and inorganic material including cell debris and mostly proteins. This is done so as to purify the sample so we are only left with DNA at the end.
The supernatant in the centrifuged tubes was then transferred to clean 2 mL collection tubes. 250 μL of Solution C2 was added to each tubes and vortexed for 5 seconds. They were then incubated at 4 C for 5 minutes in a bucket of ice. The tubes were then centrifuged at room temperature for 1 minute at 10,000 x g.
The precipitated material was disposed of as this is mostly protein and not the pure DNA we are looking for. The tubes were incubated at 4 C to precipitate out more of this "stuff" and centrifuged again for the same reason.
600 μL of the supernatant was then transferred to a clean 2 mL collection tube, careful to avoid disturbing the pellet. 200 μL of Solution C3 was added to the collection tubes and vortexed briefly. They were then incubated at 4 C for 5 minutes. The tubes were then centrifuged at room temperature for 1 minute at 10,000 x g.
MoBio says "Solution C3 is patented Inhibitor Removal Technology®(IRT) and is a second reagent to precipitate additional non-DNA organic and inorganic material including humic acid, cell debris, and proteins." Again, vortexed, incubated and centrifuged to precipitate out the impurities.
750 μL of supernatant were then transferred to a clean 2 mL collection tube, avoiding the pellet. Solution C4 was shaken and then 1.2 mL of Solution C4 was added to the supernatant and vortexes for 5 seconds.
MoBio says "Solution C4 is a high concentration salt solution. Since DNA binds tightly to silica at high salt concentrations, this will adjust the DNA solution salt concentrations to allow binding of DNA, but not non-DNA organic and inorganic material that may still be present at low levels, to the Spin Filters."
Then, 675 μL of the solution was added to a collection tube that contained a filter in the tube. This was centrifuged at room temperature for 1 minute at 10,000 x g. The flow through was then discarded and this was repeated 2 more times for each tube. 500 μL of Solution C5 was then added to the tube and centrifuged for 30 seconds at room temperature at 10,000 x g. This flow through is also discarded. It is then centrifuged at room temperature for 1 minute at 10,000 x g and flow through is discarded.
DNA in the solution binds to the silica membrane on the filer during the centrifuge process. Contaminants are then contained in the flow through which is then discarded, leaving a filter with pure DNA. Solution C5 is an ethanol based wash solution that cleans the and purifies the DNA even further. The second centrifuging for 1 minute is done to rid the filter of the residual ethanol solution.
The filter that contains pure DNA was then transferred to another 2 mL collection tube. 100 μL of Solution C6 was pipetted on the center of the filter membrane, careful NOT to touch the actual filter. This was then centrifuged at room temperature for 30 seconds at 10,000 x g. The FILTER was discarded.
Solution C6 is a sterile elution buffer that releases the DNA from the filter membrane and allows it to the flow through to the bottom of the collection tube. The resulting liquid in the tube is the pure DNA.
Note: MoBio recommends storing the DNA frozen at -20 C - -80 C.
Results
Pure DNA yo
References
HSHP Biology. DNA [Cartoon]. Accessed online 15 November 2017 at <http://hshpbiologyphoenixes.weebly.com/uploads/3/9/6/8/39688888/dna-alphabet_orig.jpg>.
MoBio Laboratories. "PowerSoil DNA Isolation Kit: Instruction Manual". MoBio Laboratories.
Thomson, Ashley. "Lab #8: DNA Isolation."
 |
| (Photo: HSHP Biology) |
Introduction
The purpose of this lab was ultimately to familiarize ourselves with the PowerSoil DNA Isolation Kit to successfully purify a DNA sample from a pure E. coli culture in LB broth.. The first step will be obtaining the cells, then through use of the kit and instructions we will break down the membrane and release the DNA from the cells. The next step will be to precipitate the DNA and finally, purify the sample. "The PowerSoil DNA is intended for use with environmental samples containing a high humid acid content including difficult soil types such as compost, sediment and manure...The isolated DNA has a high level of purity allowing for more successful PCR amplification of organisms from the sample." (MoBio) Since the sample we are using is already a pure E. coli sample, all of the purification is a tad overkill but will be done anyways according to the provided instructions. Another important thing to note is that we do not know exactly what is in the various solutions we will be using because it is proprietary knowledge, owned by MoBio Laboratories.
From MoBio Laboratories: "The PowerSoil® DNA Isolation Kit is effective at removing PCR inhibitors from even the most difficult soil types. Environmental samples are added to a bead beating tube for rapid and thorough homogenization. Cell lysis occurs by mechanical and chemical methods. Total genomic DNA is captured on a silica membrane in a spin column format. DNA is then washed and eluted from the membrane. DNA is then ready for PCR analysis and other downstream applications."owerSoil
®
DNA Isolation Kit is effective at removing PCR inhibitors from even
the most difficult soil types. Environmental samples are added to a bead beating
tube for rapid and thorough homogenization. Cell lysis occurs by mechanical and
chemical methods. Total genomic DNA is captured on a silica membrane in a spin
column format. DNA is then washed and eluted from the membrane. DNA is then
ready for PCR analysis and other downstream applications.
 |
| (Photo: Lauren Lukasik) |
 |
| (Photo: Lauren Lukasik) |
0.25 mL of the E. coli/LB broth was added to each of the two provided PowerBead Tubes that included a little bit of buffer solution and small crystals. The tubes were gently vortexed.
The buffer will help disperse the particles, begin to dissolve the humic acids (if this was a soil sample) and protect the nucleic acids from degradation. The vortexing gently mixes the solution and begins to break down the cell walls.
 |
| (Photo: Amy He) |
This initial centrifuging was the first step to precipitate out the non DNA organic and inorganic material including cell debris and mostly proteins. This is done so as to purify the sample so we are only left with DNA at the end.
 |
| (Photo: Lauren Lukasik) |
The precipitated material was disposed of as this is mostly protein and not the pure DNA we are looking for. The tubes were incubated at 4 C to precipitate out more of this "stuff" and centrifuged again for the same reason.
600 μL of the supernatant was then transferred to a clean 2 mL collection tube, careful to avoid disturbing the pellet. 200 μL of Solution C3 was added to the collection tubes and vortexed briefly. They were then incubated at 4 C for 5 minutes. The tubes were then centrifuged at room temperature for 1 minute at 10,000 x g.
MoBio says "Solution C3 is patented Inhibitor Removal Technology®(IRT) and is a second reagent to precipitate additional non-DNA organic and inorganic material including humic acid, cell debris, and proteins." Again, vortexed, incubated and centrifuged to precipitate out the impurities.
750 μL of supernatant were then transferred to a clean 2 mL collection tube, avoiding the pellet. Solution C4 was shaken and then 1.2 mL of Solution C4 was added to the supernatant and vortexes for 5 seconds.
MoBio says "Solution C4 is a high concentration salt solution. Since DNA binds tightly to silica at high salt concentrations, this will adjust the DNA solution salt concentrations to allow binding of DNA, but not non-DNA organic and inorganic material that may still be present at low levels, to the Spin Filters."
 |
| (Photo: Lauren Lukasik) |
DNA in the solution binds to the silica membrane on the filer during the centrifuge process. Contaminants are then contained in the flow through which is then discarded, leaving a filter with pure DNA. Solution C5 is an ethanol based wash solution that cleans the and purifies the DNA even further. The second centrifuging for 1 minute is done to rid the filter of the residual ethanol solution.
The filter that contains pure DNA was then transferred to another 2 mL collection tube. 100 μL of Solution C6 was pipetted on the center of the filter membrane, careful NOT to touch the actual filter. This was then centrifuged at room temperature for 30 seconds at 10,000 x g. The FILTER was discarded.
Solution C6 is a sterile elution buffer that releases the DNA from the filter membrane and allows it to the flow through to the bottom of the collection tube. The resulting liquid in the tube is the pure DNA.
Note: MoBio recommends storing the DNA frozen at -20 C - -80 C.
Results
Pure DNA yo
Discussion
While this was an easy lab with very detailed instructions, it was a time consuming lab. I don't know if we are going to do anything with the DNA we separated and purified so there's no way of knowing if we did this correctly or not. It was kind of cool though.
HSHP Biology. DNA [Cartoon]. Accessed online 15 November 2017 at <http://hshpbiologyphoenixes.weebly.com/uploads/3/9/6/8/39688888/dna-alphabet_orig.jpg>.
MoBio Laboratories. "PowerSoil DNA Isolation Kit: Instruction Manual". MoBio Laboratories.
Thomson, Ashley. "Lab #8: DNA Isolation."
Comments
Post a Comment