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. Presented by, Dr. Md. Mohiuddin Masum Guided by, Prof. Laila Anjuman Banu

3. DNA Extraction and
Quantity-Quality Check
Presented by,
Dr. Md. Mohiuddin Masum
Guided by,
Prof. Laila Anjuman Banu

4. 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

5. Extraction of DNA

6. Source of DNA

8. Basic mechanisms involved in
DNA extraction

10. Why should we extract DNA?

13. Instruments used in extraction of DNA

15. Reagents used in extraction of DNA

17. Reliaprep™ blood gDNA extraction protocol

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

20.  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

21. 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

22. 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

23. 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

24. 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

25. 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

26. Troubleshooting

27. 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

28. 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.

30. DNA quality and quantity check

31. Instruments used in assessment of
DNA quantity and quality

33. Reagents used in assessment of
DNA quantity and quality

35. DNA quality and quantity measurement
protocol for Nanodrop technique

36. 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

38. 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

39. 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 arm
For record type sample ID and click Measure on the menu bar..

41. 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

42. Troubleshooting

43. 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.

45. DNA quantity check
using flurometer

46. Instruments used in assessment of
DNA quantity

48. Reagents used in assessment of
DNA quantity

50. Why flurometer?

52. DNA quantity measurement protocol for
Qubit flurometer

53. 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.

55. 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

56. 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.

58. Dilution of amplicon

59. Why dilution is needed?

61. Principle of amplicon dilution

62. ?V1S1=VS2

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

64. V1S1 = V2S2
V1: ?
S1: 10ng/μL
V2: 10μL
S2: 2ng/μL
V1 =
V2S2
S1
V1 =
10μL X 2ng/μL
10ng/μL
V1 = 2μL

65. 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

66. Thank You