Thalita Asriandina - 1308617034 - DNA Extraction

DNA Extraction
Biologi Molekuler
THALITA ASRIANDINA (1308617934)
Blood DNA Extraction With KIT
EQUIPMENT MATERIAL
Micropipette Prewash solution
Incubator concentrate
2ml collection tube Ethanol 96 – 99%
Spin column Aliquot 200 μl of blood
Centrifuge Proteinase K solution
Vortex RNase A solution
Gloves, and Lab Coat Elution buffer (ET)
Preparation for Blood Extraction
Add Prewash solution concentrate 6ml, add Ethanol 96
– 99% 9ml, then shake (prewash solution diluted 2:3)
Add prewash solution concentrate 4ml, add ethanol
96- 99% 11 ml, the shake ( prewash solution diluted
1:3)
Blood genomic dna miniprep purification
spin kit
1. First add Aliquot 200 μl of blood in 2ml collection tube.

Add 20 μl proteinase K solution. Vortex for 10- 15 s.
After that add 20 μl RNase A solution. Vortex for 10-15
s and incubate 15 to 25 C for 2 min.
2. Add 200 μl ethanol 96-100% . load the ethanol mix
lysate on spin column provided. Spin at (10,000 rpm)
for 1 min. Add 500 μl of diluted Prewash solution.
Spin at 10,000 rpm for 1 min (remove the water).
3. Add 500 μl of diluted wash solution. Spin at 10000

rpm for 1 min (remove the water) .Add 100 μl of
Elution buffer (ET). Spin at (13,000-16,000 rpm) for
3 min to dry. After that spin at 10,000 rpm for 1 min.
Genomic DNA Extraction: Whole Blood
EQUIPMENT MATERIAL
Micropipette Whole Blood (50-200μ)
Incubator Proteinase K (20μ)
GD Column GSB Buffer (200μ)
Centrifuge Tube (1,5 Absolute Ethanol
ml) (200μ)
Centrifuge W1 Buffer (400μ)
Tube Shelf Wash Buffer (600μ)
Gloves and Lab Coat Elution Buffer (100μ)
How to Extract Genomic DNA Whole Blood
1. First transfer the whole blood to centrifuge tube and
add proteinase K. After that pippete 5-6 times then
incubate for 5 mins and add GSB buffer.
2. Shake vigorously then incubate for 5 mins, add

absolute ethanol and mix. Then Transfer to GD
column and cetrifuge for 1 mins, then add W1 buffer.
3. Add wash buffer then centrifuge for 30 secs. Then,
add elution buffer and let stand for 1 min.
4. And entrifuge for 30 secs to elute. Result: 5 μg of

high quality gDNA (260/280 of 1,8-2,0)
Extract DNA from plant with kit
MATERIAL EQUIPMET
Plant tissues (100mg) Micropipette
Liquid nitrogen Mortar and pestle

GD column
GPX1 or GP1 buffer
Scalpel
GP2 buffer
Freezer
GP3 buffer Centrifuge tube (1,5 and 2 ml)
W1 buffer Centrifuge tube shelf
Wash Buffer Elution Filter column
buffer Incubator
RNaseIce GlovesLab Coat
How to Extract DNA from plant
1. Cut up to 100 mg of plant tissue
2. Freeze with liquid nitrogen
3. Grind into a fine powder
4. Transfer powder to a 1,5ml tube
5. Add GPX1 Buffer (or GP1 depending on plant species)
6. Add RNase A
7. Vortex then incubate at 60o C for 10min
8. Add GP2 Buffer
9. Vortex then incubate on ice for 3 min
10. Place a filter column in a 2 ml tube
11. Transfer mixture to filter column
12. Centrifuge
13. Discard filter column
14. Carefully transfer supernatant to new 1,5 ml tube
15. Add a 1,5 vol of GP3 Buffer
16. Vortex immediately for 5 s
17. Place a GD Column in a 2ml tube
18. Transfer 700mikrol of mixture to the GD Column
19. Centrifuge
20. Discard the flow-through and add the rest of the mixture
21. Centrifuge
22. Discard the flow-through and add W1 Buffer
23. Centrifuge
24. Discard the flow-through and add Wash Buffer
25. Centrifuge
26. Transfer the dry GD column to a new 1,5ml tube
27. Add Elution Buffer (pre-heated to 60oC)
28. Centrifuge to elute purified DNA
29. Pure genomic
Plant Genomic DNA Isolation
EQUIPMENET MATERIAL
Micropipette Isopropyl alcohol (IPA)
Mortar and pestle Fresh plant leaf
Microfuge
Sterile distilled water
Scissor
Chloroform Isoamyl
Water bath
Microfuge tube alcohol
Centrifuge tube shelf Universal plant DNA
Petri dish extraction buffer
Incubator Pure ice
Dropper Alcohol 70%
Freezer Sterile water / TE buffer
1. First, wipe all the requirements with 70% IPA before
use and take a fresh plant leaf to petri dish.Wipe the
plant leaf with sterile distilled water, then cut 1 cm of
leaf sample and take it into mortar-pestle.
2. Take 200μl of universal plant DNA extraction buffer

and add more buffer to make final volume 500μl into
mortar and grind.
3. Add equal volume of chloroform : isoamyl alcohol
mixture (24:1) to the sample and mix. Then, centrifuge at
10.000 rpm for 10 mins. Cut the microtip using scissor
and collect upper aqueous phase in another sterile
microfuge tube.
4. Then, spin down the sample at 10.000 rpm for 10 mins

and transfer the supernatant into another sterile
microfuge tube using micropipette.
5. Take in tissue homogenate into sterile microfuge
tube using the dropper, then place the microfuge tube
into water bath and incubate at 59-60℃ for 30 mins.
6. Repeat step-5 one more time and add equal volume

of pure ice cold IPA to the collected upper phase.
Then, mix and incubate at -20℃ for 30 mins.
7. Centrifuge the tube at 10.000 rpm for 10 mins, then
discard the supernatant and collect the pellet. Next,
give alcohol 70% wash and air dry ompletely. Add
50μl of sterile water of TE buffer and tap gently to
dissolve the pellet. Then, store the DNA at low
temperature.

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DNA Extraction

1. First add Aliquot 200 μl of blood in 2ml collection tube.

3. Add 500 μl of diluted wash solution. Spin at 10000

2. Shake vigorously then incubate for 5 mins, add

4. And entrifuge for 30 secs to elute. Result: 5 μg of

Liquid nitrogen Mortar and pestle

2. Take 200μl of universal plant DNA extraction buffer

4. Then, spin down the sample at 10.000 rpm for 10 mins

6. Repeat step-5 one more time and add equal volume

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