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Team:Imperial College London/Wetlab/Protocols/Miniprep
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(New page: = Miniprep Protocol = # 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. ...)
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Revision as of 13:09, 30 September 2009
Miniprep Protocol
- 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.
- Pellet 1.5-5 ml bacteria by centrifugation at 10,000 x g for 1 min at
room temperature.
- Decant or aspirate medium and discard. To the bacterial pellet add 250ul Solution I/RNase A. Resuspend cells completely by vortexing.
Complete resuspension of cell pellet is vital for obtaining good yields.
- Add 250 ul Solution II and gently mix by inverting and rotating tube 4-6
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 in use.)
- 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.
6. CAREFULLY aspirate and add the clear supernatant to a clean Type I HiBind™ miniprep column (blue) assembled in a 2 ml collection tube (provided). Ensure that the pellet is not disturbed and that no cellular debris is carried over into the column. Centrifuge 1 min at 10,000 x g at room temperature to completely pass lysate through column. 7. Discard liquid and wash column with 500 :l Buffer HB and Centrifuge 1 min at 10,000 x g. This step ensures that residual protein contamination is removed and must be included for downstream applications requiring high quality DNA. This step can be skipped if the downstream applications don’t require high quality plasmid, such as enzyme digestion or other screening methods. 8 Discard flow-through liquid and wash the column by adding 750 :l of Wash Buffer diluted with ethanol. Centrifuge 1 min at 10,000 x g as above and discard flow-through. Note: Wash Buffer Concentrate must be diluted with absolute ethanol before use. See label for directions. If refrigerated, Wash Buffer must be brought to room temperature before use. 9. Optional step: repeat wash step with another 750 :l Wash Buffer. Page 6 of 12 10 Centrifuge the empty column for 1 min at 10,000 x g to dry the column matrix. Do not skip this step - it is critical for removing ethanol from the column. 11. Place column into a clean 1.5 ml microcentrifuge tube. Add 50 :l to 100 :l (depending on desired concentration of final product) Elution Buffer (supplied) or sterile deionized water directly onto the column matrix and centrifuge 1 min at 10,000 x g to elute DNA. This represents approximately 75-80% of bound DNA. An optional second elution will yield any residual DNA, though at a lower concentration. 12. Yield and quality of DNA: determine the absorbance of an appropriate dilution (20- to 50-fold) of the sample at 260 nm and then at 280 nm. The DNA concentration is calculated as follows: DNA concentration = Absorbance260 × 50 × (Dilution Factor) :g/ml High copy number plasmids generally yield up to 25 :g of DNA from 5 ml culture. The ratio of (absorbance260)/(absorbance280) is an indication of nucleic acid purity. A value greater than 1.8 indicates > 90% nucleic acid. Alternatively, quantity (as well as quality) can sometimes best be determined by agarose gel/ethidium bromide electrophoresis by comparison to DNA samples of known concentrations. Typically, the majority of the DNA eluted is in monomeric supercoil form, though concatamers may also be present.
