Protocol 1 – DNA Extraction Part 1

– [Instructor] Before
beginning any protocol, you want to make sure that your
workspace has a proper flow to avoid contamination
of samples or reagents. This example is set up for
a right-handed student. Tips and pipettors are on the left. Racks and samples are in the middle. And the discard bucket is on the right. As your hand moves through the flow, it never unnecessarily
crosses over the sample again. This video begins after
students have spit into the DNAgenotek collection kits, capped the tubes to release the buffer, and mixed the solution by inversion. At this point, sample
tubes should not be labeled to preserve student anonymity
during the genotyping process. Using the P1000 micro-pipettor, 500 microliters is transferred from each spit collection tube into
a new microcentrifuge tube. When pipetting large volumes,
draw up the solution slowly to avoid bubbles or
contaminating the micro-pipettor. Once the solution is transferred,
close the spit tube lid and cap the sample. Repeat this process for
all remaining samples. Prior to beginning this protocol, the heat block should
be dialed to 50 degrees and turned on by flipping the switch. If it’s heating, the light will glow red. Place samples in the heat block. This incubation must proceed
for at least 90 minutes, but it can also be left overnight. Tubes containing 20
microliters of DNAgenotek’s proprietary purifying solution. PrepIT will be provided. Once the samples have been incubated, you will transfer the
entire volume to a tube containing the purifying solution. You may notice that the solution
in the tube becomes cloudy. This is normal. Mix the solution with a vortex. To turn on the vortex, press
the switch down to Touch and turn the knob clockwise
to ensure maximum speed. Hold one tube at a time and
press the top of the black pad to initiate vibration. Repeat this process for
all remaining samples. Embed all samples in ice for 10 minutes. Impurities in the solution will be removed by centrifugation. After flipping the switch in
the back of the microcentrifuge press the Open button. Remove the metal lid inside
by pulling on the knob. Make sure that the tubes are labeled either with letters or numbers before loading them into the centrifuge. Load samples with hinges facing outward. And balance the tubes across the rotor by ensuring that all
tubes have equal volumes. Replace the metal lid and press
it until it clicks securely on to the rotor. Close the lid to the
microcentrifuge gently. Change the centrifugation
time by pressing the arrows to the desired time. In this case, five minutes. Press the arrows by the speed
to navigate to the proper RPM. In this case, 14.5 thousand RPM is the max the centrifuge can perform and is close enough to our desired speed. Press the Start button. The centrifuge will begin to spin. It is suggested that
you watch the centrifuge until it reaches maximum
speed to ensure that there are no violent vibrations, which may mean that the
tubes are unbalanced or the internal lid has come loose. If this occurs, press the
Stop button immediately. As the spin is going, obtain
four new tubes and label them. When the spin complete, the lid to the centrifuge will pop open. Remove the internal lid and
remove the samples gently to avoid disturbing the pellet. Place tubes of the same
sample in line with each other to ease the transfer of the solution. After centrifugation,
you should be able to see a white pellet that has
collected on the bottom part of the tube on the side of the hinge. This pellet contains the
impurities in the solution. The DNA is in the supernatant. Using the P1000, transfer 400
microliters of supernatant, which contains the DNA, to the new tube. Place the tip on the
opposite side of the tube from the pellet and draw up
slowly to avoid disturbing it. Repeat this process for
the remaining samples.


Add a Comment

Your email address will not be published. Required fields are marked *