Telt Method -DNA Plasmid Preperation
note: This method is "not very good" in that it does not seperate plasmid DNA from chromosomal DNA. The advantage of this is that if there is no plasmid, you will get the chromosomal DNA. Use this prep to seperate the chromosomal DNA when there is no plasmid in the cell.
- Use sterile techniques throughout
- all centrifuge steps are at room temperature
- put a tube of 100% ethanol on ice at start of procedure
- Grow cells overnight in 3mL LB (with appropriate antibiotics and sugars) in a 13 x 100mm tube at 37C (or other appropriate temperature) in a rotator.
- Pour 1.5 mL into sterile microcentrifuge tube. Centrifuge cells for 30 sec. Remove supernatent by aspiration
- Resuspend cells in 100uL TELT buffer. Vortex to resuspend pellet.
- Add 100uL 1:1 Phenol/Chloroform.
- Vortex for 15 sec. (Can leave cells for 5 minutes on th ebench).
- Centfigue for 1min. Transfer top layer to clean tube.
- Add 100uL chloroform/isoamyl (24:1)
- Vortex for 15 sec. (Can leave cells for 5 minutes on th ebench.)
- Centrifuge for 1 min.
- Transfer 75uL of the supernatent to a new microcenttrifuge tube.
- Add 200uL of 100% EtOH (cold). Mix well by inversion. Incubate at room temperature 2-10 minutes.
- Centifuge for 10 minutes.
- Pour off supernatant. Add 1mL 70% EtOH. Invert several times. Pour off liquid. Spin briefly to bring liquid to bottom of tube. Remove rest of liquid by aspiration, being careful not to disturb pellet.
- Air dry pellet (about 1/2 hour). or vacuum dry 5 minutes.
- Resuspend pellet in 30uL TE buffer.
- Use caution when using phenol. If you get any on you wash immediately with lots of water, it will turn your skin white. Eyeprotection is suggested.
Telt Buffer (Usually in 1.5 mL aliquots in -20 restriction enzyme freezer)
- 50mM Tris, pH 8.0
- 62.5mM EDTA, pH 8
- 4% Triton X-100
What you need to add:
- 5mL 1M Tris, pH 8.0
- 12.5mL 500mM EDTA, pH 8
- 31.5mL 8 M LiCl
- 4mL Triton (nanopure)
Do Not Sterilize (autoclave or filger)
DO NOT put pipettes into stock bottles, pour some into a tube for your own use.
Phenol/chloroform is in a freezer pre-made
70% ethanol is made with 700mL 100% ethanol, 300mL nanopure water.
TE buffer is in sterile DNA solutions cabinet if there is none-make up with the 100X TE stock and nanopure water, autclave in 100mL aliquotes in dilution bottles.
Procedure is from : Ausubel et al., current protocols in molecular biology.