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DNA EXTRACTION
Genomic DNA extraction was done by inorganic method from the blood samples following a
standardized protocol already published. DNA extraction was done by applying the following
methodology described below:
1. Before DNA extraction the blood samples were frozen for 24 hours at -20 °C
2. Thawing of the blood samples were done at room temperature by putting falcon
tubes containing blood samples in water bath.
3. Added T.E (Tris HCl 10mM, EDTA, 0.2mM) lysis buffer in 3 ml blood and the
contents were mixed by vortex for 2-4 minutes.
4. Centrifuged the contents at 3500 rpm for 15 minutes at 4
o
C.
5. Discarded the supernatant and broke the pellet by tapping and vertex. Then added
T.E lysis buffer.
6. Repeated the No. 4 & 5 steps, in order to bring pellet light pink.
7. Discarded supernatant and left 1 ml with lysis buffer after washing with T.E. added
45 µl of proteinase K, 100 µl of 10% SDS and 2 ml TNE (Tris HCl 10mM, EDTA
2mM and NaCl 400mM) buffer for protein digestion, shaked the contents well and
incubated at 45 °C in water bath over night for complete cellular proteins digestion.
8. Added 500µl NaCl (super saturated) in the samples after digestion, mixed the
contents and chilled for 10 minutes in refrigerator for 10 minutes.
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9. Centrifuged the contents at 4000 rpm for 15 minutes at 0
o
C for splitting the
substances in two layers. Lower layer comprising proteins and salt and DNA comes
in upper liquid layer in soluble form.
10. Shifted the supernatant with 200μl pipette in new falcon tubes.
11. Added isopropanol in equal volume, DNA threads became visible after gentle
inverting of the tubes and the contents were kept in tubes for 10 minutes at room
temperature.
12. Centrifuged the contents at 4000 rpm for 5 min at 0
o
C and discarded supernatant
with care.
13. Washed the DNA with 70% ethanol (3 ml) on shaking stand for 1-3 hours and then
centrifuged contents at 4000 rpm for 5 min at 0
o
C.
14. Discarded 70% ethanol carefully saving the DNA.
15. Washed DNA with 100% ethanol (3 ml) on shaking stand for 2-4 hours.
16. Repeated step No. 13 and 14.
17. Kept the DNA pallet for air drying in an incubator or at room temperature at 37
o
C
till the odor of ethanol ends.
18. Dissolved DNA pallet by shaking gently in diluted (low) T.E (10mM Tris HCl,
0.2mM EDTA) buffer 300-500μl as per the amount of DNA.
19. For inactivating the nucleases put the tubes in a shaking water bath for 1 hour at
70°C.
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20. Shifted dissolved DNA in 2 ml cryo preservation DNA tubes labeled on sides and
caps, stored the tubes at -20
C.
DNA ESTIMATION
Ran DNA in 1.8% agarose gel at 100 volts for 60 minutes to estimate and check its quality and
then amplified by PCR using the synthesized primers of particular gene. DNA was quantified by
using 1.8% agarose gel. Agarose gel was prepared using following methodology:
1. Dispensed agarose powder 1.8g in conical flask, added TAE 1X buffer 100 ml in the
flask and put it into microwave oven for 2 minutes.
2. Recovered the solution from oven and kept it at room temperature to allow it to cool
down.
3. Added ethidium bromide 6 μl solution (10mg/ μl) as a DNA stainer.
4. Dispensed the gel into caster. Well making combs were also put in caster. Left the gel to
solidify for 1 hour at room temperature.
Loaded 5l contents in the wells of gel including 2l of DNA and 3l loading dye (bromophenol
blue) as a tracing substance. Ran gel for 60 minutes at 100 volts. Loaded a DNA ladder (100 bp)
for reference along with DNA samples. The bands of DNA were observed in UV light
compartment. The DNA samples were quantified and brought at equal concentration of 50 ng/
μl.

Unformatted Attachment Preview

DNA EXTRACTION Genomic DNA extraction was done by inorganic method from the blood samples following a standardized protocol already published. DNA extraction was done by applying the following methodology described below: 1. Before DNA extraction the blood samples were frozen for 24 hours at -20 °C 2. Thawing of the blood samples were done at room temperature by putting falcon tubes containing blood samples in water bath. 3. Added T.E (Tris HCl 10mM, EDTA, 0.2mM) lysis buffer in 3 ml blood and the contents were mixed by vortex for 2-4 minutes. 4. Centrifuged the contents at 3500 rpm for 15 minutes at 4o C. 5. Discarded the supernatant and broke the pellet by tapping and vertex. Then added T.E lysis buffer. 6. Repeated the No. 4 & 5 steps, in order to bring pellet light pink. 7. Discarded supernatant and left 1 ml with lysis buffer after washing with T.E. added 45 µl of proteinase K, 100 µl of 10% SDS and 2 ml TNE (Tris HCl 10mM, EDTA 2mM and NaCl 400mM) buffer for protein digestion, shaked the contents well and incubated at 45 °C in water bath over night for complete cellular proteins digestion. 8. Added 500µl NaCl (super saturated) in the samples after digestion, mixed the contents and chilled for 10 minutes in refrigerator for 10 minutes. 9. Centrifuged the contents at 4000 rpm for 15 minutes at 0o C for splitting the substances in two layers. Lower layer comprising proteins and salt and DNA comes in upper liquid layer in soluble form. 10. Shifted the supernatant with 200μl pipette in new falcon tubes. 11. Added isopropanol in equal volume, DNA threads became visible after gentle inverting of the tubes and the contents were kept in tubes for 10 minutes at room temperature. 12. Centrifuged the contents at 4000 rpm for 5 min at 0o C and discarded supernatant with care. 13. Washed the DNA with 70% ethanol (3 ml) on shaking stand for 1-3 hours and then centrifuged contents at 4000 rpm for 5 min at 0o C. 14. Discarded 70% ethanol carefully saving the DNA. 15. Washed DNA with 100% ethanol (3 ml) on shaking stand for 2-4 hours. 16. Repeated step No. 13 and 14. 17. Kept the DNA pallet for air drying in an incubator or at room temperature at 37o C till the odor of ethanol ends. 18. Dissolved DNA pallet by shaking gently in diluted (low) T.E (10mM Tris HCl, 0.2mM EDTA) buffer 300-500μl as per the amount of DNA. 19. For inactivating the nucleases put the tubes in a shaking water bath for 1 hour at 70°C. 20. Shifted dissolved DNA in 2 ml cryo preservation DNA tubes labeled on sides and caps, stored the tubes at -20C. DNA ESTIMATION Ran DNA in 1.8% agarose gel at 100 volts for 60 minutes to estimate and check its quality and then amplified by PCR using the synthesized primers of particular gene. DNA was quantified by using 1.8% agarose gel. Agarose gel was prepared using following methodology: 1. Dispensed agarose powder 1.8g in conical flask, added TAE 1X buffer 100 ml in the flask and put it into microwave oven for 2 minutes. 2. Recovered the solution from oven and kept it at room temperature to allow it to cool down. 3. Added ethidium bromide 6 μl solution (10mg/ μl) as a DNA stainer. 4. Dispensed the gel into caster. Well making combs were also put in caster. Left the gel to solidify for 1 hour at room temperature. Loaded 5l contents in the wells of gel including 2l of DNA and 3l loading dye (bromophenol blue) as a tracing substance. Ran gel for 60 minutes at 100 volts. Loaded a DNA ladder (100 bp) for reference along with DNA samples. The bands of DNA were observed in UV light compartment. The DNA samples were quantified and brought at equal concentration of 50 ng/ μl. Name: Description: ...
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