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