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in this video we will demonstrate DNA
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extraction from blood using the kaya amp
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DNA mini and blood mini kit the DNA
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extraction will be carried out in
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duplicate on a single blood sample
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please note that blood sample collection
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should always be performed by a
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phlebotomist before starting your DNA
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extraction ensure that the area where
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this is being carried out is clean and
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has been decontaminated this can be done
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by spraying the bench for pets and
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equipment to be used with freshly made
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10% bleach solution allowed us to sit
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for fifteen to thirty minutes and then
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wipe away any residual bleach followed
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by rinsing with clean water and finally
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spraying with 70% ethanol gloves are to
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be worn at all times throughout the
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extraction process and change frequently
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especially if coming into contact with
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other surfaces that might be
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contaminated also check that all
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equipment and consumables you are going
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to use are in place and that the buffers
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in the kit have been prepared for
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example the appropriate volumes of
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ethanol have been added to buffers a w1
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and a w-2
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you're heating block must have been set
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at 56 degrees centigrade and ready to
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use
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before commencing with DNA extraction
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please ensure to familiarize yourself
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with the protocol so you have prepared
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the area where you will be carrying out
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your extractions and you will begin by
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labeling the micro tubes with sample IDs
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using a permanent marker we label the
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sides of our tubes as well in case Evon
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all comes into contact with the
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microtubules mix the blood by inverting
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the tube approximately 10 times to
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ensure you have a homogenous sample
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vortex the protein ASX briefly followed
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by a quick centrifugation step
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approximately 10 seconds the pet 20
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microliters of protein is K into each
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micro tube prepare 200 microliters of
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blood into each tube do this slowly as
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blood is viscous cap the blood sample
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and put to one side add 200 microliters
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of buffer al to each sample
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close the tube and vortex for about 15
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seconds and the step will be repeated
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for all subsequent samples transfer the
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micro tubes of the heating block that
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has been set at 56 degrees centigrade
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and incubate the samples for 10 minutes
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at the end of the incubation period
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remove the samples from the heating
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block and briefly centrifuge to remove
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any droplets from the lids add 200
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microliters of absolute ethanol to each
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micro tube and mix each sample by
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briefly pulse vortexing for about 15
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seconds briefly centrifuge the micro
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tubes prepare a mini spin column for
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each sample label your spin columns
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ensuring that each spin column
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corresponds to the sample ID we usually
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label the sides of the spin columns as
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well in case the ID on the lid is
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smudged by ethanol carefully transfer
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the mixture from the previous step to
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the spin column taking care not to wet
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the room of the spin column and this is
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repeated with all subsequent samples
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closed spin column and centrifuge at
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6000 G that's about 8,000 rpm for one
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minute at the end of the centrifugation
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period all the filtrate should be in the
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collection tube transfer the spin
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columns which now contain the DNA to
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clean collection tubes and discard the
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collection tubes containing the lysate
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carefully open the spin column and add
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500 microliters of aw1 buffer taking
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care not to wet the room remember to
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change your tip between each sample
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close the caps and return the samples to
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the centrifuge and centrifuge for one
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minute again at 6000 G at is 8,000 rpm
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at the end of the centrifugation period
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check that all the filtrate is in the
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collection tube if there is still some
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in the spin column just repeat that
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centrifugation step discard the
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collection tube and transfer the spin
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columns to clean collection tubes
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every open the lid and add 500
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microliters of a w-2 without wetting the
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rim of these spin columns close the cap
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and centrifuge at full speed that's
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20,000 G around 14000 rpm for three
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minutes
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discard the filtrate from the collection
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tube into a waste container as the
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collection tube is going to be reused
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dry the rim of each collection tube on a
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paper towel and replace the spin column
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centrifuge at full speed for one minute
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this ensures that all residual wash
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popper and ethanol are removed which
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could affect downstream processes label
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clean 1.5 mil micro tubes one for each
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sample and transfer the spin columns to
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each corresponding micro tube the DNA
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that was on the spin column will be
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eluted from the column to the AE buffer
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add 200 microliters of AE buffer to each
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spin column remembering to change tips
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between samples incubate for one minute
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at room temperature centrifuge for one
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minute at 6000 G that's 8,000 rpm
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really the filtrate transferring this
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back onto the spin column repeat the
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incubation and centrifugation steps this
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repeat dilution step optimizes a DNA
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yield for each sample this completes
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your DNA extraction of your blood
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samples and you will now evaluate the
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DNA concentration and quality for this
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we are going to perform a couple of
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quality control steps we recommend that
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you remove an aliquot of each sample for
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carrying out your QC s a total of five
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microliters will be sufficient while
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leaving the rest of your samples are
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nice we are going to use the nanodrop
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spectrophotometer to determine the DNA
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concentration and purity this will be
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followed by gel electrophoresis to
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establish DNA integrity for the nanodrop
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we will use 1.5 microliters of DNA
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sample and we'll use the AE buffer to
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blank the instrument because this is
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what the DNA samples have been diluted
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in samples are always fun down to ensure
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that the DNA is at the bottom of the
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tube a controlled DNA sample is always
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included of known concentration this is
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used to check that the instrument is
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working optimally in assessing the DNA
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while the concentration and the two 6280
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ratios are important it is also critical
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that the two 6230 ratios are good the
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two 6230 ratios should be between 1.5
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and 3 when reviewing the results we can
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see that one of the samples hasn't
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performed optimally while the other one
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has if the sample was to be used for the
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genotyping FA we will be carrying out it
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would require an ethanol precipitation
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step to purify that sample
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the next thing we are going to do is to
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assess the DNA integrity and the samples
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will be run on a 1% agarose gel loading
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buffer is added to each sample including
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the DNA ladder which will be used to run
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alongside the samples running buffer in
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this case 1 times tbe
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containing a 30 embroid is added to the
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tank the gel is lowered into the gel
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tank and the samples together with the
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high molecular weight DNA ladder are
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loaded onto the gel the gel is said to
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run at 100 volts for 20 to 30 minutes at
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the end of the run time the gel is
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removed from the gel tank and
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transferred to a gel dock system or
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transilluminator to visualize the DNA as
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you can see from these results the
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samples are all of high molecular weight
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this gel picture contains samples that
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are both of high molecular weight DNA as
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well as degraded DNA we will now
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demonstrate how to dry down DNA samples
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using the DNA stable plate this would
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enable the transportation of DNA at
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ambient temperature this needs to be
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performed in a PCR hood or a laminar
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flow turn on the pcr hood or laminar
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flow at least 5 minutes before starting
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aliquot the same volume of DNA across
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the plate the positions of each DNA
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sample has to be recorded accurately
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using a sample recording template
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containing a plate layout the plate will
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remain in the PCR hood or the laminar
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flow until the samples had dried down
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completely the drying time will depend
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on the volume of DNA used approximate
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drying times can be found in the DNA
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stable protocol however the drying time
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should be monitored and the final time
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recorded the PCR hood or laminar flow
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should be kept running for the duration
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of the drying downtime when all the DNA
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plates are completely dried down seal
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the plate with a PC
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our plate seal ensuring that all wells
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are completely airtight this is going to
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be achieved by using a seal applicator
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close the plate each plate has a lid and
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place a DNA stable plate into a shipping
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bag place the bag and accompanying
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documentation for example the sample
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submission forms export license details
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into a shipping box
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to summarize you have been shown how to
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extract DNA from whole blood or the
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quality control checks entail drying
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down DNA in a DNA stable plate and
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packaging of the plate with the dried
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down samples for shipment
