http://www.protocol-online.org/prot/Molecular_Biology/DNA/DNA_Extraction___Purification/DNA_Extraction_from_Bacteria_and_Other_Organisms/index.html

Many or most protocols for isolating DNA from E. coli are focused on getting a high yield of plasmid or phage DNA with minimal bacterial genome (main chromosome) DNA, because we most often are looking to harvest a large quantity of a gene we have cloned into the plasmid or phage.   So be sure to look for protocols for genomic DNA and not plasmid mini-prep protocols.

http://molecularcloning.com/index.php?prt=3

 THANK YOU

Dr. Brian Thomas Foley. I am sure to get my results from it.

What type of sample are you isolating from? A pure culture? Environmental? Blood? That will make a difference in the best protocol to use.

 Cassandra Vogel Kotter, Thanks for pointing towards the critical error. Can I know for PURE CULTURE of E. Coli, stored at -80 temperature, which protocol You suggest

That should be pretty easy then, since there aren't a lot of contaminants. E.coli is also quite easy to lyse, so you can probably skip any of the fancy lysis steps, like proteinase K, etc... What are the downstream processes you plan to use the DNA for (i.e. pure enough to sequence or run qPCR or just use for cloning)? Are you open to using a DNA extraction kit or are you trying to make all the reagents yourself?

Cassandra Vogel Kotter

I am open to use extraction kit, though I want to learn it by conventional technioque. After cloning, my aim is to go for protein expression. Can You advance Your suggestions.

See: He, F. (2011). E. coli Genomic DNA Extraction. Bio-protocol Bio101: e97. DOI: 10.21769/BioProtoc.97.

https://bio-protocol.org/bio101/e97#biaoti1285

or

Green MR, Sambrook J: Isolating DNA from Gram-negative bacteria. Cold Spring Harb Protoc 2017, 2017:pdb.prot093369.

Here is the protocol we use:

- Resuspend pellet of 50 mL culture in 23 mL TE (0.15 M NaCL, 0.1 M EDTA, 10 mM Tris pH8)

- Add 2 mL 25% SDS

- Incubate with mixing at 60 °C for 10 minutes.

- Cool to room temperature.

- Add 2 mg Proteinase K

- Incubate for 30 minutes at 37 °C

- Add 0.5 mL 5 M NaClO4

- Extract DNA by Phenol/Chloroform Extraction: Add 1 volume Phenol/chloroform (50/50) Mix delicately for approx. 5 minutes

Centrifuge for 10 mins. at 3000 rpm

Remove upper aqueous phase (use a cut tip)

Repeat extraction process until white interface (contaminants) is removed

- Very slowly, add 2 volumes cold absolute ethanol to 1 volume upper aqueous phase.

- Remove genomic DNA by 'spooling' with a glass rod.

- Dry in laminar flow hood

- Resuspend the 'spooled' genomic DNA in approx. 400 µL TE + RNAse (5 mg in 50 mL TE).

Pls find :

Single protocol for extraction of gDNA from bacteria and yeast.

Laurie Vingataramin & Eric H. Frost.

biotechniques 58:120-125 (March 2015). Doi 10.2144/000114263