DNA Extraction andQuantity-Quality Check

Presented by,Dr. Md. Mohiuddin Masum

Guided by,Prof. Laila Anjuman Banu

Objectives Explain the basic mechanisms involved in DNA

extraction Describe the steps involved in gDNA extraction

from blood Explain the processes involved in quality and

quantity check of extracted DNA using Nanodrop technique

Decribe the steps of quantity check of amplicon using flurometer

Decribe the principle of dilution of amplicon

Extraction of DNA

Source of DNA

Basic mechanisms involved inDNA extraction

Why should we extract DNA?

Instruments used in extraction of DNA

Reagents used in extraction of DNA

Reliaprep™ blood gDNA extraction protocol

Thaw the frozen blood completely

Thoroughly mix the blood sample for at least 10 minutes by vortexing at room temperature

20 μl protein-ase K solution with yellow

200 μl blood with barrier tips and briefly mix

200 μl cell lysis buffer with blue tips

Take a 1.5 ml micro-centrifuge tube and using micropipette add

Close the cap of tube and mix by vortexing for at least 10 seconds

Incubate at 56°C for 10 minutes in a heating block

After removing the tube from heating block, add 250 μl of binding buffer and mix by vortexing for 10 seconds

Place a binding column into an empty collection tube

Add the contents of the tube to the binding column using micropipette with barrier tips

Close the cap of the column and place it in a microcentrifuge machine and centrifuge for 1 minute at 14,000 rpm speed

Check the binding column to make sure the lysate has completely passed through the membrane. If lysate is still visible on top of the membrane, centrifuge the column for another minute

Remove the collection tube containing flow through, and discard the liquid as hazardous waste

Place the binding column into a fresh collection tube. Add 500μl of column wash solution using micropipette with blue tips to the column, and centrifuge for 3 minutes at 14,000 rpm.

Discard the flowthrough and repeat this step twice for a total of three washes

Place the binding column in a clean 1.5ml microcentrifuge tube. Add 50 μl TE buffer and centrifuge for 1 minute at 14,000 rpm

Keep the extracted DNA at 4 °c for 24 hour then check the quality and quantity of the extracted DNA according to protocol in Nanodrop.

After quality and quantity check keep extracted DNA at -26 °c

Troubleshooting

If blood forms clots

Blood was not sufficiently mixed

For good lysis, the blood must be mixed prior to adding proteinase K and lysis buffer

If wash buffer do not pass through the column

Samples were not centrifuged long enough. Recentrifuge for 1 minute

Or, Centrifuge was not generating sufficient

gravitational force

Blood gDNA Miniprep system and columns are designed for use with a microcentrifuge capable of generating at least 12,000 rpm.

DNA quality and quantity check

Instruments used in assessment ofDNA quantity and quality

Reagents used in assessment ofDNA quantity and quality

DNA quality and quantity measurement protocol for Nanodrop

technique

Clean the Nanodrop with nuclease free water and then wipe the water by a dry lint-free Kimwipes tissue

Repeat this step twice for a total of three washes

Open the nanodrop software and select nucleic acid from the menu bar.

Select DNA from the right side of the menu bar

Raise the sampling arm and load 1.5 μl blank (TE buffer) on lower measurement pedestal then bring down the sampling arm

Click blank to measure and store the reference spectrum

Wipe the blank from both measurement pedestal surfaces with a dry tissue

Short spin the microcentrifuge tube containing extracted DNA samples in the spinner

Raise the sampling arm and load 1.5 μl sample onto the lower measurement pedestal and bring down the sampling armFor record type sample ID and click Measure on the menu bar..

Wipe the sample from both measurement pedestal surfaces with a dry tissue

Then go Reports from left side of the menu bar and click on Export and select location to save the values in Excel form

Clean the Nanodrop as the 1st step

Finally click exit to come out

Troubleshooting

If DNA yield is low Blood contained low levels of leukocytes

White blood cell levels less than 4 × 106 per milliliter will give reduced yields.

Blood was too old.

Best yields are obtained with fresh blood.

DNA quantity checkusing flurometer

Instruments used in assessment ofDNA quantity

Reagents used in assessment ofDNA quantity

Why flurometer?

DNA quantity measurement protocol for Qubit flurometer

Take two assay tubes for the standards and one tube for each sample

Prepare the “Working Solution” by diluting the Qubit™reagent 1:200 in Qubit™ buffer. Prepare 200 μL of Working Solution for each standard and sample.

Prepare the assay tubes-

Standardassay Tubes

User Sampleassay Tubes

Volume of Working Solution 190 μL 180-199 μLVolume of standard 10μL —Volume of user sample to add — 1-20μLTotal volume in each assay tube 200 μL 200 μL

Vortex all tubes for 2–3 seconds

Incubate the tubes for 2 minutes at room temperature

Insert the tubes in the fluorometer and take readings.

Dilution of amplicon

Why dilution is needed?

Principle of amplicon dilution

?V1S1=VS2

V1S1 = V2S2V1: Desired sample volume

S1: Desired sample conc.

V2: PCR product volume

S2: PCR product conc.

V1S1 = V2S2

V1: ?S1: 10ng/μL

V2: 10μLS2: 2ng/μL

V1 =V2S2S1

V1 = 10μL X 2ng/μL10ng/μL

V1 = 2μL

V1: 2μL

PCR Product to be taken: 2μL

Nuclease free water to be taken: 8μL

Desided 10μL of PCR product with DNA conc.

2ng/μL

Thank You