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| | + | {{Imperial/09/TemplateTop}} |
| | + | {{Imperial/09/Tabs/Main/Wetlab/Protocols}} |
| | = Miniprep Protocol = | | = Miniprep Protocol = |
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| - | # Inoculate 5 ml LB/ampicillin (50 ug/ml) medium placed in a 10-20 ml culture tube with E.coli carrying desired plasmid and grow at 37'C with agitation for 12-16 h.
| + | Protocol is from the E.Z.N.A HP Plasmid Mini Kit I Spin Protocol. |
| - | # Pellet 1.5-5 ml bacteria by centrifugation at 10,000 x g for 1 min at
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| - | room temperature.
| + | A pdf of the protocol can be found [http://www.genex.cl/stock/EZNAD694401.pdf here] |
| - | # Decant or aspirate medium and discard. To the bacterial pellet add 250ul Solution I/RNase A. Resuspend cells completely by vortexing.<br><br>
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| - | Complete resuspension of cell pellet is vital for obtaining good yields.
| + | {{Imperial/09/TemplateBottom}} |
| - | # Add 250 ul Solution II and gently mix by inverting and rotating tube 4-6
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| - | times to obtain a cleared lysate. Leave for 2 min at room temperature to incubate. Avoid vigorous mixing as this will shear chromosomal DNA and lower plasmid purity. (Store Solution II tightly capped when not
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| - | in use.)
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| - | # Add 350 ul Solution III and gently mix by inverting several times until a flocculent white precipitate forms. Centrifuge at 10,000 x g for 10 minutes at room temperature.
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| - | 6. CAREFULLY aspirate and add the clear supernatant to a clean Type
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| - | I HiBind™ miniprep column (blue) assembled in a 2 ml collection tube
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| - | (provided). Ensure that the pellet is not disturbed and that no cellular
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| - | debris is carried over into the column. Centrifuge 1 min at 10,000 x g at
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| - | room temperature to completely pass lysate through column.
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| - | 7. Discard liquid and wash column with 500 :l Buffer HB and Centrifuge 1 min
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| - | at 10,000 x g. This step ensures that residual protein contamination is
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| - | removed and must be included for downstream applications requiring high
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| - | quality DNA. This step can be skipped if the downstream applications
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| - | don’t require high quality plasmid, such as enzyme digestion or other
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| - | screening methods.
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| - | 8 Discard flow-through liquid and wash the column by adding 750 :l of Wash
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| - | Buffer diluted with ethanol. Centrifuge 1 min at 10,000 x g as above and
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| - | discard flow-through.
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| - | Note: Wash Buffer Concentrate must be diluted with absolute ethanol before use. See
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| - | label for directions. If refrigerated, Wash Buffer must be brought to room temperature
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| - | before use.
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| - | 9. Optional step: repeat wash step with another 750 :l Wash Buffer.
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| - | Page 6 of 12
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| - | 10 Centrifuge the empty column for 1 min at 10,000 x g to dry the column
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| - | matrix. Do not skip this step - it is critical for removing ethanol from
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| - | the column.
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| - | 11. Place column into a clean 1.5 ml microcentrifuge tube. Add 50 :l to
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| - | 100 :l (depending on desired concentration of final product) Elution
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| - | Buffer (supplied) or sterile deionized water directly onto the column
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| - | matrix and centrifuge 1 min at 10,000 x g to elute DNA. This
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| - | represents approximately 75-80% of bound DNA. An optional second
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| - | elution will yield any residual DNA, though at a lower concentration.
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| - | 12. Yield and quality of DNA: determine the absorbance of an appropriate
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| - | dilution (20- to 50-fold) of the sample at 260 nm and then at 280 nm. The
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| - | DNA concentration is calculated as follows:
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| - | DNA concentration = Absorbance260 × 50 × (Dilution Factor) :g/ml
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| - | High copy number plasmids generally yield up to 25 :g of DNA from 5 ml
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| - | culture. The ratio of (absorbance260)/(absorbance280) is an indication of
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| - | nucleic acid purity. A value greater than 1.8 indicates > 90% nucleic acid.
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| - | Alternatively, quantity (as well as quality) can sometimes best be
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| - | determined by agarose gel/ethidium bromide electrophoresis by
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| - | comparison to DNA samples of known concentrations. Typically, the
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| - | majority of the DNA eluted is in monomeric supercoil form, though
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| - | concatamers may also be present.
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