This is a cached page for the URL (http://axon.med.harvard.edu/~cepko/protocol/mike/D2.html). To see the most recent version of this page, please click here.
Protocol Online is not affiliated with the authors of this page nor responsible for its content.
About Cache
Protocol D

Protocol D.2

Quick DNA Plasmid Prep.

 

This is a very fast mini-prep protocol which is suitable for sequence analysis and restriction digests. Although the yield is higher than Protocol D.1, there is considerable chromosomal DNA, RNA and protein contamination.

 

Solutions

Sucrose/Tris

25% sucrose 25 g sucrose

50 mM Tris pH 8.0 5 ml 1M Tris pH 8.0

up to 100 ml with Q

store at room temperature

 

Triton Lysing Mix

5% Triton X-100 5 ml Triton X-100

5% sucrose 5 g sucrose

50 mM Tris pH 7.5 5 ml 1M Tris pH 7.5

50 mM EDTA 10 ml 0.5 M EDTA pH 8.0

up to 100 ml with Q

store at room temperature

 

2.5 M KOAc

2.5 M postassium acetate 24.5 g potassium acetate

pH to 4.8 with glaicial acetic acid

up to 100 ml with Q

store at room temperature

 

 

 

 

 

Procedure

 

 

• Pellet 1.5 ml of an overnight culture in an eppendorf tube by spinning at14K. Resuspend in 15 ml Sucrose/Tris.

Prepare Lysozyme Mixture (10mg/ml (Sigma #L6876) in Sucrose/Tris)

Prepare boiling water bath

 

• Add 100 ml Triton Lysing Mix and briefly vortex on half speed. Add 10 ml Lysozyme Mixture, invert, and incubate at room temperature for 5'.

 

• Boil for 4' and spin at 14K for 15' in the cold room.

 

• Remove the pellet with a toothpick and add 12 ml 2.5 M KOAc and 100 ml isopropanol; incubate at -80o for 10'.

 

• Spin 10' and wash the pellet with 80% EtOH.

 

• Dry and resuspend in 30 ml Q.

 

 

I use 5 ml for a standard digest (RNaseA is required), and 13.5 ml for sequencing (Protocol S.1).