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I.
Purpose: To extract genomic DNA
from Mouse tail and other types of animal tissues
II.
Chemical Principal: Modified
Proteinase K - phenol extraction method.
III.
Pretreatment
of Tissue Samples:
A. Mouse tail and Ear
1.
Place 0.5-1.0cm
mouse tail (10-20mg) or 1/3 to a whole of mouse ear (5-20mg) into 96
deep
well plate(s).
2.
Load the sample
plates on the AutoGenprep 965/960
3.
Run [Digest] Protocol*. The AutoGenprep 965/960 adds 0.15ml of Reagent M2
(Tissue Digestion
2) and 0.15ml of Reagent M1 (Tissue Digestion 1), containing the
pre-dissolved Proteinase K at
the concentration of 0.4 - 1.0mg/ml** into the plates and mixes them.
4. Remove
the sample plate from the 965/960, seal the plate and incubate overnight at 60-65°C.
* Step 3 and 4 can be done manually.
**
Prepare solution for every run by dissolving appropriate aliquots of
ProK provided in 965/960
kit with each aliquot of Reagent M1. Standard concentration
for overnight digestion of
mouse tail is 0.4mg/ml. For other types of tissues or quicker
digestion, a higher
concentration of ProK solution may be required.
B. Mouse Liver and Kidney
1.
Place mouse liver or kidney into appropriate tubes, and add equal amount
of Reagent M2
(Tissue Digestion 2) and Reagent M1 (Tissue Digestion 1),
containing the pre-dissolved
ProK at the concentration of 0.4 - 1.0mg/ml** for a final
concentration of 15 - 60mg tissue/ml.
2.
Incubate the samples overnight at
60-65°C.
3.
Transfer 0.3ml (equivalent to
5-20mg tissue) of Proteinase K digests to 96 well deep well plates.
IV. Protocol Parameters:
A.
Sample Volume: 0.3ml
of Proteinase K digest (equivalent to 5-20mg of tissues)
B.
Maximum Number of Samples:
384 samples (4 plates) per run.
C.
Processing Time: 2.5
hours for 192 samples (2 plates)
4.0 hours for 384 samples (4 plates)
This includes a 20 minutes drying period
D.
Yield: 10 - 40
mg§
of genomic DNA (10-30mg of mouse tail / 1/3
to a whole mouse ear)
10 - 30
mg§
of genomic DNA (5 -20mg of mouse liver or
kidney)
§ The actual yield may vary depending on condition and volume of
starting samples.
E.
Quality: OD260/280
values are 1.65 - 1.85
OD230/260 values
are <0.5
The DNA can be used directly in downstream applications such as
fluorescent
DNA sequencing, PCR, restriction enzyme digestions and
more.
V.
Running the Protocol:
A.
Load Reagents and Sample Plates
B.
Select [Extraction] Protocol
C.
Enter Number of Samples
D.
Start the Run
VI. Example of the extracted
genomic DNA on the AutoGenprep 965/960:
Gel Electrophoresis
of the extracted genomic DNA
Gel: 0.7% agarose, TBE
Condition: 50V x 45 min.
Sample: 0.5µl
M: l/HindIII
marker
VII. Extraction Process:
| Process Site |
Purpose |
System Process |
1. Automated
or Manual |
Digest Tissues |
Dissolve Pro K with Reagent M1, and
add this combined solution with Reagent
M2 to samples, and incubate overnight at
at
60-65°C. |
| 2. Automated |
Remove RNA |
Add Reagents R8 and mix. |
| 3. Automated |
Remove Protein and
Cellular Debris |
Add Reagent R3, mix,
centrifuge to
pellet debris and transfer supernatant to
new (DNA) plate. |
| 4. Automated |
Precipitate DNA |
Add Reagent R4, mix,
precipitate
DNA, centrifuge and discard
supernatant. |
| 5. Automated |
Wash DNA |
Add Reagent R5/6/7, mix,
centrifuge, and
discard supernatant, repeat. |
| 6. Automated |
Dry DNA |
Dry DNA by heating. |
| 7. Automated |
Resuspend DNA |
Add Reagent R9 and mix. |
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