Plasmid Preparation

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Revision as of 15:42, 12 June 2006

This is the procedure we tried for plasmid preparation of pSB1A3:

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 ultrapure water

CTAB: 5% CTAB in ultrapure 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.)

(Preparation):

Label 2 Eppendorf tubes per sample, turn heat block on to 100°C or prepare boiling waterbath, prepare fresh lysozyme, put CTAB(5%) in warm waterbath with stirring to make sure crystals completely dissolved. Carry out the procedure without refridgeration until you get to rid of the CTAB in step 11 to prevent the CTAB from dropping out of solution and not precipitating the plasmid.

Procedure

Step 1: Set up a 3-5mL overnight in a 20mL sterilin culture tube with appropriate selection – remembering to inoculate with a single colony.

Step 2: Spin down cells (enough to give cell pellet–- remove supernatent (clear, yellow)

Step 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)

Step 4: When all samples are re-suspended add 20ul 50mg/mL lysozyme (store on ice until use)

Step 5: Invert until well mixed, and incubate @ room temperature until 5 minutes after the addition of lysozyme, or until viscosity increases markedly (no more than 10 minutes), pierce lids

Step 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!).

Step 7: Spin down immediately for 15 minutes at full speed (cell pellet contains chromosomal DNA); move supernatent to clean tube

Step 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.

Step 9: 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. This may improve yield because CTAB itself precipitates in the absence of DNA at lower temperatures (about 20°C)

Step 10: Spin for 3 minutes at full-speed

Step 11: Remove supernatent completely, but do not let samples dry out

Step 12: Resuspend each sample with CTAB-DNA with 300ul 1.2M NaCl (may require vortex)

Step 13: Add 780ul ‘absolute’ ethanol to each sample, mix by inversion until mixing is complete, and spin for 10 minutes at full-speed.

Step 14: Remove supernatent and wash pellet with 70% ethanol (breaking up pellet)

Step 15: Spin-down washed pellet for 5 mins at full speed, and remove ethanol wash

Step 16: Let remaining 70% ethanol evaporate (ie. Air-dry), to dryness (may take an hour or more on a cold day).