DNA extraction from Blood

Summary

This video tutorial guides viewers through the process of DNA extraction from blood using the QIAamp DNA Mini and Blood Mini Kit. It covers preparation, including bench decontamination and equipment setup, and the extraction procedure in duplicate. The video also demonstrates quality control steps like DNA concentration and integrity assessment using a spectrophotometer and agarose gel electrophoresis. Finally, it shows how to dry down DNA samples for ambient temperature transport using a DNA stable plate, ensuring safe and efficient sample shipment.

Takeaways

• 🔬 The video demonstrates DNA extraction from blood using the QIAamp DNA Mini and Blood Mini Kit.
• 🩸 DNA extraction is performed in duplicate on a single blood sample, emphasizing the importance of sample consistency.
• 🧼 A clean and decontaminated workspace is crucial, with a detailed procedure for bench and equipment disinfection provided.
• 🧤 Gloves must be worn at all times and changed frequently to prevent cross-contamination.
• 📝 Proper labeling of micro tubes with sample IDs is essential for tracking and organization.
• 🌡️ A heating block set at 56 degrees Celsius is used for incubation, indicating temperature control is a critical step.
• 🧪 The use of specific buffers and reagents is detailed, highlighting the importance of following the kit's protocol.
• 🌀 Vortexing and centrifugation steps are integral to the process, ensuring thorough mixing and separation of components.
• 🧬 The integrity and concentration of the extracted DNA are assessed using a NanoDrop spectrophotometer and gel electrophoresis.
• 🔎 The video provides a method for drying down DNA samples using a DNA stable plate for easy transportation and storage.
• 📦 The final steps include packaging the dried DNA samples for shipment, with attention to documentation and proper sealing.

Q & A

What is the purpose of the video?

The purpose of the video is to demonstrate the process of DNA extraction from blood using the QIAamp DNA Mini and Blood Mini Kit.

Who should perform the blood sample collection?

Blood sample collection should always be performed by a phlebotomist.

What is the first step in preparing the area for DNA extraction?

The first step is to clean and decontaminate the area by spraying the bench and equipment with a 10% bleach solution, allowing it to sit for 15-30 minutes, then wiping and rinsing with clean water, and finally spraying with 70% ethanol.

Why is it important to wear gloves during the extraction process?

Gloves are to be worn at all times to prevent contamination of the samples and to protect the person handling the samples.

What should be done to the buffers in the kit before starting the DNA extraction?

The appropriate volumes of ethanol should be added to buffers AW1 and AW2 before starting the DNA extraction.

At what temperature should the heating block be set for the DNA extraction process?

The heating block must be set at 56 degrees centigrade for the DNA extraction process.

How is the blood sample prepared before adding the buffer?

The blood sample should be mixed by inverting the tube approximately 10 times and then vortexed briefly followed by a quick centrifugation step.

What is the purpose of incubating the samples at 56 degrees centigrade?

Incubating the samples at 56 degrees centigrade helps in lysing the cells and breaking down the proteins to release the DNA.

How are the spin columns prepared for DNA extraction?

Spin columns are prepared by labeling them with corresponding sample IDs and ensuring they are ready for the transfer of the mixture from the previous step.

What is the purpose of the AW1 and AW2 buffers in the DNA extraction process?

AW1 buffer is used to wash the DNA away from contaminants, while AW2 buffer is used to further purify the DNA by removing any remaining contaminants and salts.

How is the DNA eluted from the spin column?

The DNA is eluted from the spin column by adding AE buffer to the column and then centrifuging, which transfers the DNA into the corresponding micro tube.

What quality control steps are recommended after DNA extraction?

Quality control steps recommended include determining DNA concentration and purity using a nanodrop spectrophotometer and assessing DNA integrity through gel electrophoresis.

Why is it necessary to dry down DNA samples using the DNA stable plate?

Drying down DNA samples using the DNA stable plate enables the transportation of DNA at ambient temperature without the need for cold storage.

Outlines

00:00

🧬 DNA Extraction Preparation and Procedure

This section of the video script outlines the initial steps for DNA extraction from blood using the Kaya Amp DNA Mini and Blood Mini Kit. It emphasizes the importance of a clean and decontaminated workspace, with specific instructions on how to prepare the area with bleach and ethanol. The necessity of wearing gloves and ensuring all equipment and consumables are ready is also highlighted. The protocol involves labeling micro tubes, mixing blood samples, adding protein ASX and buffer AL, and incubating samples at 56 degrees Celsius. The process continues with the use of mini spin columns for DNA purification, and the addition of AW1 and AW2 buffers for washing. The paragraph concludes with the transfer of DNA to clean collection tubes for further processing.

05:00

🔬 DNA Quality Control and Integrity Assessment

Paragraph 2 details the quality control steps following DNA extraction. It describes the process of using a nanodrop spectrophotometer to measure DNA concentration and purity, with an emphasis on the importance of the A260/230 ratios. The paragraph also covers the use of gel electrophoresis to assess DNA integrity, including the preparation of samples and the use of a DNA ladder for comparison. The video script mentions the potential need for ethanol precipitation to purify samples that do not meet quality standards. Additionally, it demonstrates how to dry down DNA samples using a DNA stable plate for transportation at ambient temperature, including the steps for recording sample positions and ensuring complete drying before sealing and packaging the plate for shipment.

10:03

📦 DNA Sample Drying and Packaging for Shipment

The final paragraph of the script focuses on the final steps of DNA sample processing for shipment. It explains how to aliquot DNA samples across a DNA stable plate and record their positions accurately. The importance of drying the samples completely in a PCR hood or laminar flow is highlighted, along with the monitoring of drying times. Once dried, the samples are sealed with a PCR plate seal to ensure airtight conditions. The script concludes with instructions for packaging the sealed plate along with necessary documentation into a shipping box, summarizing the entire process from DNA extraction to the preparation of samples for shipment.

Keywords

💡DNA Extraction

DNA Extraction refers to the process of separating DNA from other cellular components, such as proteins and lipids. In the video, DNA extraction is the primary focus, demonstrating how to extract DNA from blood using specific kits. The process is crucial for various molecular biology applications, including genetic testing and forensic analysis.

💡Kaya Amp DNA Mini and Blood Mini Kit

The Kaya Amp DNA Mini and Blood Mini Kit is a commercial product used in the video for DNA extraction from blood samples. These kits typically contain all the necessary reagents and instructions to perform DNA extraction efficiently. The use of such kits in the video highlights the importance of standardized protocols in laboratory procedures.

💡Phlebotomist

A phlebotomist is a healthcare professional trained to draw blood from patients. The video emphasizes that blood sample collection should be performed by a phlebotomist, indicating the need for trained personnel to ensure the quality and safety of blood samples used for DNA extraction.

💡Decontamination

Decontamination is the process of removing or killing contaminants, such as microorganisms, from surfaces or objects. In the script, the area where DNA extraction is carried out must be decontaminated to prevent contamination of samples. This step is critical in maintaining the integrity of the DNA and the accuracy of the results.

💡Ethanol

Ethanol is a solvent used in the DNA extraction process to precipitate DNA and remove contaminants. The script mentions adding ethanol to buffers and using it in the washing steps of the extraction process. Ethanol's ability to denature proteins and precipitate nucleic acids makes it a common component in molecular biology protocols.

💡Proteinase K

Proteinase K is a proteolytic enzyme used to break down proteins, which helps in the release of DNA from cells. In the video, proteinase K is vortexed and centrifuged to ensure proper mixing with the blood sample, facilitating DNA extraction. Its use is a standard procedure in many DNA extraction protocols.

💡Spin Column

A spin column is a device used in molecular biology for separating and purifying substances based on their size, often through a membrane. In the video, spin columns are used to bind and wash DNA, removing unwanted contaminants. The use of spin columns is a common technique in DNA purification processes.

💡Buffer AW1 and AW2

Buffers AW1 and AW2 are components of the DNA extraction kit used in the video. These buffers are used in the washing steps to further purify the DNA. AW1 is typically a low-salt buffer, while AW2 is a high-salt buffer, each serving to remove different contaminants and improve DNA purity.

💡Nanodrop Spectrophotometer

The Nanodrop Spectrophotometer is an instrument used to measure the concentration and purity of nucleic acids, such as DNA. In the video, it is used to assess the DNA concentration and purity after extraction. This step is crucial for ensuring the quality of the DNA samples for subsequent applications.

💡Agarose Gel Electrophoresis

Agarose gel electrophoresis is a technique used to separate DNA fragments by size based on their migration through an agarose gel matrix under an electric field. In the video, this method is used to assess the integrity of the DNA. The presence of high molecular weight DNA bands on the gel indicates successful extraction and minimal degradation.

💡DNA Stable Plate

A DNA Stable Plate is a device used for drying down DNA samples, which allows for the transportation of DNA at ambient temperatures without the need for cold storage. In the video, DNA samples are dried in a DNA stable plate for shipment, highlighting a practical aspect of handling and storing DNA samples for further analysis.

Highlights

Demonstration of DNA extraction from blood using Kaya amp DNA mini and blood mini kit.

DNA extraction is performed in duplicate on a single blood sample.

Blood sample collection should be done by a phlebotomist.

The workspace must be clean and decontaminated before starting the DNA extraction.

Use of 10% bleach solution and 70% ethanol for workspace disinfection.

Gloves must be worn and changed frequently to prevent contamination.

All equipment and consumables must be prepared and ready before extraction.

Buffers in the kit must have appropriate volumes of ethanol added.

Heating block must be set at 56 degrees centigrade for sample incubation.

Labeling of micro tubes with sample IDs is essential for organization.

Blood must be mixed by inverting the tube to ensure a homogenous sample.

Protein ASX is vortexed and centrifuged briefly before adding to micro tubes.

Buffer AL is added to each sample and vortexed for proper mixing.

Samples are incubated in a heating block at 56 degrees centigrade for 10 minutes.

Absolute ethanol is added to each micro tube and mixed after incubation.

Mini spin columns are prepared and labeled for each sample.

Mixture from previous step is carefully transferred to the spin column.

Spin columns are centrifuged at 6000 G to separate DNA from other components.

AW1 buffer is used to wash the DNA in the spin column.

AW2 buffer is used for a second wash to ensure purity of the DNA.

DNA is eluted from the spin column using AE buffer.

DNA concentration and purity are determined using a nanodrop spectrophotometer.

Gel electrophoresis is performed to establish DNA integrity.

High molecular weight DNA is visualized on the gel.

Demonstration of drying down DNA samples using a DNA stable plate for transportation.

DNA stable plate is sealed and packaged for shipment after drying.

Transcripts

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