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I.
Purpose: To
extract plasmid, cosmid, BAC or PAC DNA from cultures of E.
coli.
II.
Chemical
Principal: Modified
alkaline lysis - phenol extraction method.
III.
Protocol
Parameters:
A.
Sample
Volume: 0.2 - 1.8
ml liquid culture of E. coli.
Note:
We assume bacterial culture, the density of which is 4.0-7.0
at OD660, may be used. The appropriate starting volume
depends on cell density, copy number and so forth. It is
important to optimize the culture conditions and starting volume
to obtain high yield and high quality of DNA.
B.
Maximum
Number of Samples: 384
samples (4 plates) per run.
C.
Processing
Time: 2.2 hours for 192 samples (2 plates)
3.7 hours for 384
samples (4 plates)
This includes a 25
minute drying period
D.
Yield:
5.0 - 7.0 mg
of purified Plasmid DNA per 1.0 ml culture of plasmid.
0.3-0.6
mg
of purified BAC DNA per 1.0 ml culture of BAC.
Note:
The actual yield may vary depending on cell density and other
variable factors.
E.
Quality:
Typical OD260/280 values are 1.75 -
1.85.
The DNA can be used directly in downstream applications such as
fluorescent
DNA sequencing, PCR, Southern blotting and restriction
endonuclease
digestions.
IV.
Running the Protocol:
A. Load
Reagents and Samples
B. Select
a Protocol
C. Enter
Number of Samples and Starting Volume
D. Start
the Run
V.
Example
of the extracted DNA on the AutoGenprep 965/960:
A.
Extracted Plasmid DNA:
Gel electrophoresis
of the extracted Plasmid DNA
(0.7% agarose gel,
100V)
M: Hind III
marker
Lanes 1-12: Extracted
DNA on the AutoGenprep 965/960
Lanes a-d: Manually
extracted DNA
B.
Extracted BAC DNA:
Gel electrophoresis
of extracted and
digested BAC samples
(0.7%, 100V)
M:
l/Hind
III.
1-4:
Extracted BAC on the AutoGenprep 965/960
a-d: Manually
extracted BAC
1= Intact BAC, 2 = NotI
digest, 3 = EcoRI digest and
4 = HindIII
digest
VI.
Extraction
and Purification Process:
| Process Site |
Purpose |
System Process |
| 1. Automated |
Concentrate Cells |
Centrifuge overnight
culture. Discard supernatant. |
| 2. Automated |
Lyse Cells, Remove RNA |
Add Reagents R1 and R2
and
mix. |
| 3. Automated |
Remove Protein
and Cellular Debris |
Add Reagent R3, mix,
centrifuge to pellet debris, transfer supernatant to
new plate, discard debris. |
| 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 |
Evaporate Alcohol |
Dry DNA on heating
station. |
| 7. Automated |
Resuspend DNA |
Add Reagent R9 and mix. |
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