DNA Extraction using Reliaprep™ blood gDNA extraction protocol DNA quantity-quality check using Nanodrop and Amplicon quantity check using Fluorometer DNA extraction procedure video YouTube link https://www.youtube.com/watch?v=uk2H4tJUAto Nanodrop procedure video YouTube link https://www.youtube.com/watch?v=JlMuF0FAU1g Fluorometer procedure video YouTube link https://www.youtube.com/watch?v=To1vSP1bNxo

1. DNA Extraction and
Quantity-Quality Check
Dr. Md. Mohiuddin Masum
Bangabandhu Sheikh Mujib Medical University
Dhaka, Bangladesh

2. 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

3. Extraction of DNA

4. Source of DNA

6. Basic mechanisms involved in
DNA extraction

8. Why should we extract DNA?

11. Instruments used in extraction of DNA

13. Reagents used in extraction of DNA

15. Reliaprep™ blood gDNA extraction protocol

17. Thaw the frozen blood completely
Thoroughly mix the blood sample for at least 10 minutes by
vortexing at room temperature

18.  20 l protein-ase K solution with yellowμ
 200 l blood with barrier tips and briefly mixμ
 200 l cell lysis buffer with blue tipsμ
Take a 1.5 ml micro-centrifuge tube
and using micropipette add

19. 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 l ofμ
binding buffer and mix by vortexing for 10 seconds

20. 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

21. 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

22. Place the binding column into a fresh collection tube. Add
500 l of column wash solution using micropipette with blueμ
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

23. Place the binding column in a clean 1.5ml microcentrifuge
tube. Add 50 l TE buffer and centrifuge for 1 minute atμ
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

24. Troubleshooting

25. 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

26. 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.

27. DNA extraction procedure video YouTube link
https://www.youtube.com/watch?v=uk2H4tJUAto

28. DNA quality and quantity check

29. Instruments used in assessment of
DNA quantity and quality

31. Reagents used in assessment of
DNA quantity and quality

33. DNA quality and quantity measurement
protocol for Nanodrop technique

34. 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

36. Raise the sampling arm and load 1.5 l blank (TE buffer) onμ
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

37. Short spin the microcentrifuge tube containing extracted
DNA samples in the spinner
Raise the sampling arm and load 1.5 l sample onto the lowerμ
measurement pedestal and bring down the sampling arm
For record type sample ID and click Measure on the menu bar..

39. 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

40. Troubleshooting

41. 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.

42. Nanodrop procedure video YouTube link
https://www.youtube.com/watch?v=JlMuF0FAU1g

43. DNA quantity check
using flurometer

44. Instruments used in assessment of
DNA quantity

46. Reagents used in assessment of
DNA quantity

48. Why flurometer?

50. DNA quantity measurement protocol for
Qubit flurometer

51. 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 L of Workingμ
Solution for each standard and sample.

53. Prepare the assay tubes-
Standard
assay Tubes
User Sample
assay Tubes
Volume of Working Solution 190 μL 180-199 μL
Volume of standard 10μL —
Volume of user sample to add — 1-20μL
Total volume in each assay tube 200 μL 200 μL

54. 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.

55. Dilution of amplicon

56. Why dilution is needed?

58. Principle of amplicon dilution

59. ?V1S1=VS2

60. V1S1 = V2S2
V1: Desired sample volume
S1: Desired sample conc.
V2: PCR product volume
S2: PCR product conc.

61. V1S1 = V2S2
V1: ?
S1: 10ng/ Lμ
V2: 10 Lμ
S2: 2ng/ Lμ
V1 =
V2S2
S1
V1 =
10 L X 2ng/ Lμ μ
10ng/ Lμ
V1 = 2 Lμ

62. V1: 2 Lμ
PCR Product to be taken: 2 Lμ
Nuclease free water to be taken: 8 Lμ
Desided 10 L ofμ
PCR product
with DNA conc.
2ng/ Lμ

63. Fluorometer procedure video YouTube link
https://www.youtube.com/watch?v=To1vSP1bNxo

64. Thank You