Download BioTeke Corporation Technical Manual

Note: for laboratory research use only.
Plant DNA Rapid Extraction Kit
(Spin-column)
A kit for the isolation of genomic DNA from plants
Cat. #. DP3111（50 preps）
DP3112（100 preps）
DP3113（200 preps）
BioTeke Corporation
I. Kit Content, Storage and Stability
Content
Buffer P1
Buffer P2
Buffer P3
RNase A
Buffer WB
Storage
RT
RT
RT
-20℃
RT
50 preps
100 preps
200 preps
（DP3111）
（DP3112） （DP3113）
30 ml
60 ml
120 ml
7 ml
14 ml
28 ml
50 ml
100 ml
200 ml
200μl
400 μl
800 μl
15 ml
25ml
25ml
Add the ration ethanol before use.
15 ml
20 ml
40 ml
50
100
200
Buffer EB
RT
Separation Column
RT
A
Spin-column AC
RT
50
100
Collection Tube
RT
50
100
（2ml）
Reagents, when stored properly, are stable for 12 months.
200
200
Note：
1) Dilute Buffer WB with four volume absolute ethanol before starting.
2) Buffer P1 and Buffer P3 may form precipitation due to low storage temperature.
If necessary, incubate buffers by at 37°C until until clear. Cool to room
temperature before use.
3) Please ensure the bottles of buffers tightly capped when not in use, preventing
reagents evaporating, oxidation and pH change.
II. Principle
Dry or fresh plant tissues are grinded and then lysed in a special buffer containing
detergent. Proteins, polysaccharides, and cellular debris are subsequently precipitated.
Contaminants are further removed by isopropanol precipitation of DNA. Binding
conditions are then adjusted. The sample is then applied to a spin-column and
centrifuged. DNA binds to the silicified membrane while contaminants such as
proteins and polysaccharides are efficiently removed by two-step wash. Purified DNA
is eluted in a small volume of low ionic strength buffer or water.
III. Features
1. Rapid, DNA isolation under 60 min.
2. Stable, high-quality silicified membrane and ideal buffer system ensure the
reproducible
results.
3. High purity, purified DNA typically has an A260/A280 ratio between 1.7 and 1.9.
4. Isolated DNA ranges from 30Kb to 50Kb and can be directly used for most
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downstream applications, including PCR, Southern-blot, Restriction digestion
reactions, etc.
IV. Notes
1.
Please read this section before starting.
All the centrifugation steps can be performed at room temperature.
2.
Prepare the β- mercaptoethanol by yourself.
3.
Set water bath to 65℃.
4.
Buffer P3 contains the stimulating compounds. Please wear latex gloves, avoiding
skin, eyes and cloth to be contaminated. If that, please wash with water or
5.
physiological saline.
No EDTA in Buffer EB, which will have no influence on down-stream reactions.
Also you can use water when eluting, but please ensure PH>7.5 and store at -20℃. If for long-term storage, dissolve DNA in TE (10mM Tris-HCl, 1mM
EDTA, pH 8.0). Because EDTA will affect the down-stream reactions, dilute the
solution before use.
V. Procedure
Dilute buffer WB with four volume absolute ethanol before starting.
Pre-warm Buffer P1 to 65℃ and add β-mercaptoethanol at the final
concentration 0.2%.
1. Pre-warm buffer P1, mortars, pestles and sterilized water to 65℃
2. Take proper plant tissue to mortar grind in liquid nitrogen.
3. Transfer powders (fresh plant tissue 100mg or gross weight tissue 30mg) to a 1.5ml
centrifuge tube and add 550μl pre-warm Buffer P1(added β-mercaptoethanol to final
concentration 0.2%) and 4μl RNaseA. Vortex for 1min and let it sit at room
temperature for 10min.
4. Add 130μl Buffer P2 and mix thoroughly. Centrifuge at 12,000rpm for 3min.
5. Carefully transfer the supernatant to a Separation column, centrifuge at 12,000rpm
for 1min, and collect the flow through.
6. Add 1.5 volumes of Buffer P3 to the flow through and mix thoroughly.
7. Place a Spin-column AC to a collection tube. Transfer the mixture (including
precipitate) to the Spin-column AC. Centrifuge at 12,000rpm for 1min. Discard the
flow through.
8. Add 700μl Buffer WB (check if ethanol added!). Centrifuge for 1min at 13,000rpm.
Discard the flow-through.
9. Add 500μl buffer WB. Centrifuge for 1min at 13,000rpm. Discard the flow-through
10. Then centrifuge the empty Spin-column AC at 13,000rpm for 3-5 min.
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11. Transfer the Spin-column AC to a clean 1.5ml microcentrifuge tube, add 50μl Buffer
EB (warm in 65-℃70℃ before use) directly onto the silicified membrane. Incubate
3-5 min at room temperature. Centrifuge at 13,000rpm at 1 min.
The volume of elution buffer could be adjusted according to needs.
Appropriately reduce elution volume can increase concentration. But the
minimum volume is 50µl; too less will decrease the elution efficiency and the
DNA yield.
12. Keep DNA at 2-8℃ (-20℃ for long term storage).
VI. Troubleshooting
Problem
Low DNA yield
RNA contamination
No Product
DNA colored
Possible causes
Sample excess or
incomplete lysis
RNA rich in plant
Not add ethanol to Buffer
WB
Not enough wash times
Too much material
Low DNA elution
A260 too high
DNA digestion
inhibition
Ethanol residues in spin
column or collection tube
bottom.
Use water or other
solution replace buffer EB
Silicified membrane
eluted, influence A260
value.
Silicified membrane
eluted, inhibited
digestion.
Ethanol residues in Spincolumn or collection tube
bottom.
Advices
Use proper amount sample
and sufficiently grinded.
Add 8μl RNase instead 4μl
after step 3
Add the ration ethanol
before use.
Add 500μl WB or 100%
ethanol to wash after step 7
Reduce material, don’t
exceed
Ensure do step 10, or affect
the elution efficiency.
Please reading carefully step
10, just use Buffer EB
Centrifuge at 13,000 rpm for
1 minutes, carefully use the
supernatant.
Centrifuge at 13,000 rpm for
1 minutes, carefully use the
supernatant.
Ensure do step 10 and air dry
for a moment.
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