TRY THESE LINKS

https://link.springer.com/protocol/10.1385/1-59259-409-3%3A79?no-access=true

http://bio-protocol.org/e30

the conjugation may be mechanically interrupted at different times by vigorous agitation of the mixture, just may be

https://www.ncbi.nlm.nih.gov › NCBI › Literature › PubMed Central (PMC)

[Bio101] Plasmid DNA Extraction from E. coli Using Alkaline Lysis Method

Microbiology > Microbial genetics > DNA

Molecular Biology > DNA > DNA extraction

Bacteria > Escherichia > Escherichia coli > DNA

Author: Fanglian He

2/5/2011, 87250 views, 31 Q&A

DOI: https://doi.org/10.21769/BioProtoc.30

[Abstract] This is a quick and efficient way to extract E. coli plasmid DNA without using commercial kits.

Materials and Reagents

RNAase (Life Technologies, Invitrogen™)

Isopropanol (EM Science)

Ethanol (VWR Chemical)

Tryptone

Yeast extract

NaCl

Glucose

EDTA

0.2 N NaOH

SDS

KOAc

Potassium acetate

Glacial acetate

Tris-HCl (pH 8.0)

Luria-Bertani broth (LB) medium: Bacto-tryptone (BD Biosciences), yeast extract (BD Biosciences) (see Recipes)

Resuspension solution (P1 buffer) (see Recipes)

Lysis solution (P2 buffer) (see Recipes)

Neutralizing solution (P3 buffer) (see Recipes)

TE (see Recipes)

Equipment

Table-top centrifuges

1.5-ml eppendorf tube

37 °C heat blocker

Procedure

Grow bacterial (E. coli) culture in LB medium with appropriate antibiotics at 37 °C overnight (O/N) with shaking. For >10 copies plasmid, 3 ml cell culture is usually enough.

Transfer O/N culture to a 1.5-ml eppendorf tube, and spin down cell culture (twice) at high speed for 1 min at table-top centrifuge.

Discard the supernatant. To remove the liquid completely by upside down tube onto a piece of paper towel for a few sec.

Add 100 μl of resuspension solution (P1 buffer) into each tube, and vortex to completely resuspend cell pellet

Add 100 μl of lysis solution (P2 buffer) and mix by gently inverting the tube 5-6 times. The solution should quickly turn transparent and become more viscous indicating bacterial

lysis has taken place.

Add 150 μl of neutralizing solution (P3 buffer) and mix by inverting the tubes several times. At this point bacterial chromosomal DNA is usually seen as a white precipitate.

Centrifuge the tubes at high speed for 10 min.

Carefully transfer the supernatant (try to not disturb the white precipitate) to a new labeled 1.5-ml eppendorf tube with a 1ml pipette.

Add 2.5-3 volume of 200-proof cold ethanol (stores at -20 °C) to each tube and mix by inverting the tubes a few times.

Spin down plasmid DNA precipitate (transparency pellet) at high speed for 10 min.

Discard the supernatant and remove the remaining liquid as much as possible by leaving the tube upside-down on a piece of paper towel, then keep the tubes in a tube holder and air dry for 10-20 min. To dry faster, keep tubes at 37 °C heat blocker. DNA precipitate turns white when dry.

Resuspend the DNA pellet with 50 μl TE. Completely dissolve the pellet by pipetting solution several times.

Note: Large amounts of RNA is present in the DNA sample. Therefore, for subsequent reactions, for example, to digest plasmid DNA, add 1-5 μl (1 mg ml-1) RNAase to the digestion solution to completely remove RNA. Or, add RNAase directly to the resuspension solution with a final concentration of 1 mg ml-1.

Recipes

LB medium

1% Tryptone

0.5% yeast extract

200 mM NaCl

Resuspension solution (P1 buffer)

50 mM glucose

10 mM EDTA

25 mM Tris (pH 8.0)

Store at 40 °C.

Lysis solution (P2 buffer)

0.2 N NaOH

1% SDS

Store at room temperature.

Neutralizing solution (P3 buffer)

3 M KOAc (pH 6.0)

For 100 ml solution, 60 ml 5 M potassium acetate (49.07 g potassium acetate in 100 ml H2O)

11.5 ml glacial acetate and 28.5 ml H2O, store at room temperature.

TE

1 mM EDTA

10 mM Tris-HCl (pH 8.0)

Note: P1, P2, P3 buffers from the QIAGEN DNA extraction kit also work well.

Thank you so much for your considerations and help. I will try it again by paying attention to your recommends. Thank you again.

Aylin - your question is somewhat confusing. If you are doing conjugation then you don't need to isolate the plasmid but work on your conjugation method. If you are talking about transformation then you would need to purify the plasmid. However this is likely to be effective only for smaller plasmids, so it depends upon what plasmid you are working with. If you wanted to provide more details you might get more precise answers.

 Thank you for your correcting, Dr. Benedik. I am sorry for imperfect knowledge in my question. Now, I have realized that. On the other hand, I have overcome the problem about my research. Thank you so much again for your consideration.