The document details a protocol for DNA extraction using the phenol-chloroform method, outlining the materials and step-by-step procedures for handling blood samples and isolating DNA. It specifies necessary reagents, equipment, and various steps including lysis, centrifugation, and purification processes over two days. The final goal is to obtain high-quality DNA for further analysis such as quantification and genotyping.

1. DNA extraction
Dr.Gopisankar.M.G.
Junior Resident
Department of Pharmacology
JIPMER
Mr. Ravi Prasad
PhD Scholar
Department of Pharmacology
JIPMER
Phenol Chloroform Method of DNA extraction
© JIPMER , Puducherry

2. ‘Knowledge not shared is wasted’
- Clan Jacobs

3. Things to be kept ready – for 20 samples
• Polypropylene tube – 20 +20 + 20 = 60 Tarson tubes
• RBC lysis solution – (10 ml ×20 ×4) around 2L
• WBC lysis solution – ( 2.25ml × 20) around 200 mL
• SDS -10% Sodium dodecyl sulphate – (125μL × 20) prepare at least
10 mL
• Proteinase K (10mg/mL) – (50μL × 20) (1ml, prepare at least 10 mL)
• Milli Q water ( 75μL×20) – Keep around 10 mL

4. • Saturated NaCl – need (1ml × 20 ) = 20 mL
• Chloroform – (3.5mL ×20) = 1 bottle
• Equilibrated phenol – (2.5ml ×20) = 1 bottle
• Chloroform : Octanol (ratio 24:1) (2.5mL ×20)= 100 ml
• Ice cold ethanol – 2 bottles
• 70% ethanol – 1.5ml(if you take 1.5ml centrifuge tube) ×20 = 30 mL
• TE buffer = (200μL×20)  5mL

5. DAY 1

6. • Collect 5 ml blood in a polypropylene tube containing 100μl of 10%
EDTA disodium salt
• Centrifuge at 2500 rpm for 10 min at 40C
• Remove the plasma and discard

12. RBC lysis solution
Add 10 ml of RBC lysis solution .Mix for 3 min

35. • If heme remains it will affect the quality of the DNA

36. WBC solution

40. & Mix gently

45. •Add 125 μl of SDS

46. •Add 50 μl of Proteinase K

47. Add 75 μl of milliQ water

48. Mix the solution Gently

50. INCUBATE @ 370C
over night

51. END of DAY 1

52. Day 2

53. Check
• Whether lysis is complete
• If it is complete the solution will be homogenous and it will freely
moves when tilted to and fro
• If lysis incomplete  invert the tube for 10 times gently and incubate
them @ 37C for 4 hours

54. Complete lysis

55. Add 1ml of saturated NaCl and mix using Pasture pipette

56. Mix with pasture pipette

57. Chloroform

58. Add 3.5 ml of chloroform

59. Mix gently
• Mix till it becomes
homogenous mixture
• The aqueous volume and
chloroform volume must
be equal

60. Centrifuge @ 4000 rpm @ 40C for 20 min

61. Mark  next set of test tubes

62. Open and keep like this for easiness of transfer

63. Keep test tubes side by side & transfer

64. Eg. From 13 take the supernatant(avoid taking
the brownish layer) to the A13

65. Equilibrated phenol

66. • Add 2.5 ml of equilibrated phenol
• Mix gently for 5 min

67. Mix gently for 5 min

68. Centrifuge @4000 rpm for 20 min at 40C

69. Take upper layer without disturbing and shift
to new set of tubes

70. • Mark the next set of
tubes
• To which supernatant is to
be added

71. • Again keep side by side and transfer the
supernatant like this

72. Transfer up to this level only

73. Add 2.5 ml Chloroform : Octanol (24:1)

74. Mix gently for 5 min

75. Centrifuge at 4000 rpm for 20 min at 40C

76. Centrifuged sample

77. Mark the next set of test
tubes !!!

78. Transfer the supernatant by keeping it side by side

79. Add 2 volumes of ice cold absolute ethanol

80. Mix by inverting the tubes many times

81. High molecular DNA will precipitate at this step

82. Now we have to transfer the DNA to centrifuge tube

83. • Arrange centrifuge
tubes
• In two sets
• One for 70%
Ethanol
• One for TRIS buffer

84. Centrifuge tubes arranged in stands

85. 70% ethanol

86. Pour 70% ethanol into the tubes

87. Pour TRIS buffer into the other set

88. Remove DNA using a clean pipette

89. Wash it with 70% Ethanol by tapping the tube 5- 6 times

90. Air dry the DNA

91. After drying put it in TRIS buffer (200μL of PH 7.4)
carefully and close it (DNA would dissolve in it )

92. Cover each tube with Aluminum foil

93. Incubate at 370C for over night

94. Incubate ……

95. Hm… now I have to quantify n genotype !!!!!!