Editing Plasmid Miniprep using CTAB (section)

== Plasmid DNA CTAB Miniprep from E. coli Preparation ==

<br/>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 bathwith 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 realizedthat 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 refrigerationuntil you get rid of the CTAB in step 11.&nbsp;<br/>Procedure<br/>1. Set up a 3-5mL overnight in a 20mL sterile culture tube with appropriate selection –remembering to inoculate with a single colony.&nbsp;<br/>2. Spin down cells (enough to give cell pellet, but not so much that it is difficult to resuspend) –remove supernatant(clear, yellow)&nbsp;<br/>3. Re-suspend each sample of cells in 0.4 mL STET (8% sucrose w/v, 0.1% v/v Triton X-100, 50mM EDTA, 50mM Tris pH 8.0)&nbsp;<br/>4. When all samples are re-suspended add 20ul 50 mg/mL lysozyme (store on ice until use)&nbsp;<br/>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&nbsp;<br/>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!).&nbsp;<br/>7. Spin down immediately 15 minutes at full speed (cell pellet contains chromosomal DNA), move supernatantto clean tube&nbsp;<br/>8. Add 20ul CTAB (5% w/v) to each sample, and mix by inversion 

Final CTAB concentration 20*5/(440+20)=0.217%

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)&nbsp;<br/>9. Spin for 3 minutes at full-speed&nbsp;<br/>10. Remove supernatant completely, but do not let samples dry out&nbsp;<br/>11. Resuspend each sample with CTAB-DNA with 300ul 1.2M NaCl (may require vortex)&nbsp;<br/>12. Add 780ul ‘absolute’ ethanol to each sample, mix by inversion until mixing is complete.&nbsp;<br/>13. Spin10 minutes at full-speed.&nbsp;<br/>14. Remove supernatantand wash pellet with 70% ethanol (breaking up pellet)&nbsp;<br/>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 ormore on a cold day)