CTAB DNA Extraction from E coli by bnWs0X

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									Plasmid DNA CTAB Miniprep from E. coli
    Preparation
         Label 2 Eppendorf tubes per sample, turn heat block on to 100°C or prepare boiling water bath, prepare fresh
         lysozyme, put CTAB(5%) in warm water bath with stirring to make sure crystals completely dissolved. This
         protocol works fine in the summer, then I discovered it was less good during the colder months – until I
         realized that the CTAB was dropping out of solution instead of precipitating the plasmid. The best way to
         avoid this is to carry out the procedure without refrigeration until you get rid of the CTAB in step 11.

    Procedure
             1.    Set up a 3-5mL overnight in a 20mL sterile culture tube with appropriate selection – remembering
                   to inoculate with a single colony.
             2.    Spin down cells (enough to give cell pellet, but not so much that it is difficult to resuspend) –
                   remove supernatant (clear, yellow)
             3.    Re-suspend each sample of cells in 0.4mL STET (8% sucrose w/v, 0.1% v/v Triton X-100, 50mM
                   EDTA, 50mM Tris pH 8.0)
             4.    When all samples are re-suspended add 20ul 50mg/mL lysozyme (store on ice until use)
             5.    Invert until well mixed, and incubate at room temperature until 5 minutes after the addition of
                   lysozyme, or until viscosity increasing markedly (no more than 10 minutes), pierce lids
             6.    Boil samples on heat block for exactly 45 seconds (must be about 100°C), can use boiling water
                   bath if large number of samples!) – I find a boiling water bath is better than a heating block (and
                   quicker to heat up!).
             7.    Spin down immediately 15 minutes at full speed (cell pellet contains chromosomal DNA), move
                   supernatant to clean tube
             8.    Add 20ul CTAB (5% w/v) to each sample, and mix by inversion – if successful, a white precipitate
                   will immediately form, and you should see this hanging in suspension in the sample. Incubate at
                   40°C for 5 minutes without shaking to maximize yield. If there is none visible in a particular
                   sample, then no plasmid will be recovered. I think this improves yield because CTAB itself
                   precipitates in the absence of DNA at lower temperatures (about 20°C)
             9.    Spin for 3 minutes at full-speed
             10.   Remove supernatant completely, but do not let samples dry out
             11.   Resuspend each sample with CTAB-DNA with 300ul 1.2M NaCl (may require vortex)
             12.   Add 780ul ‘absolute’ ethanol to each sample, mix by inversion until mixing is complete.
             13.   Spin 10 minutes at full-speed.
             14.   Remove supernatant and wash pellet with 70% ethanol (breaking up pellet)
             15.   Spin-down washed pellet 5 min. at full speed, and remove ethanol wash
             16.   Let remaining 70% ethanol evaporate (i.e. Air-dry), to dryness (may take an hour or more on a cold
                   day)



    Solutions
         STET

                   8% sucrose w/v
                   0.1% v/v Triton X-100
                   50mM EDTA
                   50mM Tris pH 8.0

         Lysozyme

                   50 mg/ml lysozyme (Sigma, chicken egg white) in ultra-pure water
        CTAB

                 5% CTAB in ultra-pure water
                 Precipitates at cool temperature, therefore heat and stir before use
                 Note that CTAB (Cetyltrimethylammonium bromide) is also known as Cetrimide and its proper
                 chemical name is hexadecyltrimethylammonium bromide

Notes

        This is not mine, but it seems to work well.

        None of this protocol need be done on ice. The subsequent miniprep should not contain nuclease activity. An
        RNase step can be added, 1 microlitre of a 10mg/mL solution of RNase can be added after the boiling
        treatment, with an incubation for 10 minutes at 65°C. RNase treatment risks the introduction of co-purified
        DNases. I usually don't bother with RNase treatment, instead, I add it to restriction digests or sequencing
        reactions as a very dilute solution - with no apparent problems. This boiling miniprep is the alternative to the
        alkaline lysis method of plasmid mini-prep, both of which are plasmid purification methods which can be
        called plasmid mini-prep or plasmid miniprep, plasmid mini-preparation, plasmid minipreparation, plasmid
        isolation, and erroneously as mini-plasmid preparation or mini plasmid prep. Since the method is of a
        miniature scale, it is a mini-prep - but can be used for both large and small plasmids. (In my hands upto
        20kb).

Reference

        Adapted from "A one-tube plasmid dna mini-preparation suitable for seqeuencing". Delsal G, Manifioletti G,
        Schneider C. Nucleic acids research 16 (20): 9878-9878 Oct 25 1988.

								
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