Introduction
DNA
isolation is the process in which we can purify a DNA sample using a
combination of physical and chemical methods. In this lab, we focused on a
processes called PowerSoil DNA Isolation Kit to layout a specific procedure
base to help isolate genomic DNA from the environmental sample one’s heart
desires. The kit is used specifically to tackle high humic acid content
including difficult soil types such as compost, sediment, and manure.
We happened
to work with a pure culture of E. Coli,
a collected activated sludge sample and also a pure culture of a Methylobacterium. We then just isolated
the three types of samples to where we can store the Polymerase Chain Reaction
(PCR) to use in another lab. This process is a breakthrough for genetic
modification in the science world.
Methods/Procedure
Let us
begin with simply taking the designated sample that was given (ours was the Methylobacterium) and add a fourth of a
gram to the PowerBead Tubes provided. Gently vortex for maximum mixing power.
Next, check for precipitates in your C1 solution, if theres solids then heat at
60 degrees’ Celsius till dissolved. We can now add 60 μL of Solution C1, then
Mix with shaker briefly. Now bust out the vortex and give
the O’sample a shaking for 10 minutes at maximum speed. We now can use the best
thing ever, The Centrifuge. Centrifuge tubes at 10,000 x g for 30 seconds at
room temperature. Now after 30 seconds has passed, transfer the supernatant to
a clean 2 mL Collection Tube which should be provided. We can now add 250 μL of
Solution C2 and vortex for 5 seconds. Incubate at 4°C for 5 minutes. Well, back
to the Centrifuge, keeping in mind that the tubes should be at room temperature
for 1 minute at 10,000 x g. Avoiding the pellet, transfer up to, but no more
than, 600 μl of supernatant to a clean 2 ml Collection Tube which again is
provided. Add 200 μl of Solution C3 and vortex briefly. Incubate at 4°C for 5
minutes. Centrifuge the tubes at room temperature for 1 minute at 10,000 x g. Avoiding
the pellet, transfer up to, but no more than, 750 μl of supernatant into a
clean 2 ml in provided Collection Tube. Shake that sample like you’ve never
done before to mix Solution C4 before use. Add 1200 μl of Solution C4 to the supernatant
and vortex for 5 seconds. Load approximately 675 μl onto a Spin Filter and centrifuge
at 10,000 x g for 1 minute at room temperature. Discard the flow through and
add an additional 675 μl of supernatant to the Spin Filter and centrifuge at
10,000 x g for 1 minute at room temperature. Load the remaining supernatant
onto the Spin Filter and centrifuge at 10,000 x g for 1 minute at room
temperature. A total of three loads for each sample processed are required. Add
500 μl of Solution C5 and centrifuge at room temperature for 30 seconds at
10,000 x g. Discard the flow through. Centrifuge again at room temperature for
1 minute at 10,000 x g. Carefully place spin filter in a clean 2 mL Collection
Tube (provided). Avoid splashing any Solution C5 onto the Spin Filter. Add 100
μl of Solution C6 to the center of the white filter membrane and Centrifuge at
room temperature for 30 seconds at 10,000 x g. Discard the Spin Filter. The DNA
in the tube is now ready for any downstream application. No further steps are
required. If not using immediately, store at -80 degrees’ C.
Results
We have no
results just yet for this lab due to it leading to another set of labs.
Discussion
We now can
see that I typed the word Centrifuge 1 too many times. But this was a
interesting lab that was simple but intriguing. We can use the DNA that was
isolated for PCR in a later lab.
No comments:
Post a Comment