I am trying to isolate plasmid from wild type bacteria and having problems in purification. First of all do they degenerate more rapidly and secondly are there any protocols for purification from other contaminents like RNA and chromosomal DNA (no kits).

Since I do not know what type of bacteria you are using, have a look into this protocol section.
You can find several protocols.

DNA Extraction Protocols.


[Edited to fix link]

From E. coli? On what scale?

1) what strain of bacteria are you using?

2) is this a plasmid you transfected into the basteria or an endogenous plasmid from the bacteria you are studying?

3) Are you using a kit to pruify the plasmid or are you doing caesium chloride-ethidium bromide density gradient cetrifugation?

holeung said:

From E. coli? On what scale?

Yes, on large scale

frasermoss said:

1) what strain of bacteria are you using? 2) is this a plasmid you transfected into the basteria or an endogenous plasmid from the bacteria you are studying? 3) Are you using a kit to pruify the plasmid or are you doing caesium chloride-ethidium bromide density gradient cetrifugation?

1- E.coli

2- endogenous, from wild strain
3- no kits and no gradient centrifugation.

I am using alkaline lysis method and purifying by Licl and sod. acetate/ethanol precipitation.

I dont think LiCl and NaOAc is going to cut it for your purposes. You need to isolate your plasmid from chromosoaml DNA and other contaminants and concentrate the plasmid fraction.

You should use one of the many commercially available maxiprep kits from companies like Qiagen, Sigma or Promega.

Personally I use the qiagen kits.
For large scale preps check out

http://www1.qiagen.com/Products/Plasmid/LargeScaleKits.aspx

this will help you pick the right kit for your job.

If you have not used them before they may well look expensive, but in terms of time, effort, yeild and reduced exposure to harmful reagents you just cant beat them.

Ah - I just saw the no kits in brackets.

Then your main alternative has to be CsCL EtBR density gradient centrifugation.

There are very clear protocols in the bible of Molecular Biology - otherwise known as

Molecular Cloning - A Laboratory Manual

by Joseph Sambrook, David W. Russell
ISBN: 0879695773
www.MolecularCloning.com

The only thing about CsCl purification is that it takes so very long to do and all that nasty EtBr sloshing around.

Why wont/cant you use a kit? Is the plasmid expected to be over 15kb or something?

Not all strains of bacteria are good for isolating DNA.
Take BL21DE3 (used commonly for protein expression): you will not be able to isolate DNA from that.

As "frasermoss" already asked, is that a plasmid you have transformed into the bacteria?
You should consider transfecting it into XL1 blue or a different strain used for plasmid isolation.

This protocol can be used for large scale as well

http://wheat.pw.usda.gov/~lazo/methods/lazo/dnageneral.html

Well thanks everybody but the problem is I do not have an high speed centrifuge and justification for buying kits is out of question in my working conditions (do not ask why). The plasmid is from a wild strain, about 4kb in size and isolated by alkaline lysis. Now can some one suggest a purification method.

Not my method, so no promises it will work for you, but something I found that you might be able to try that does not require anion exchange columns or an ultra centrifuge. They used a Sorvall GSA or SLA-3000.

DNA Plasmid Maxiprep Protocol

1. Pick single colony and inoculate 250 ml of LB broth containing 100 mg/l ampicillin or appropriate antibiotic. Shake at 250 RPM overnight.

2. Centrifuge cells at 5 k × g for 10 minutes.

3. Resuspend cell pellet in 5 ml of GTE buffer (50 mM Glucose, 25 mM Tris-Cl, 10 mM EDTA, pH 8) by pipetting up and down with a 10ml pipette. Optional: add a spatula tip of lysozyme powder. A good suspension is consistent without clumps of cell pellet.

4. Add 10 ml of NaOH/SDS lysis solution (0.2 M NaOH, 1% SDS). Use a spatula to stir and dissolve the cells until the solution becomes clear, yellow. Alternatively, you can shake the bottle for 5 seconds.

5. Quickly add 7.5 ml of 5 M potassium acetate solution (pH 4.8). This solution neutralizes NaOH in the previous lysis step while precipitating the genomic DNA and SDS in an insoluble white, rubbery precipitate. Shake bottle thoroughly. Centrifuge at 10 k × g for 10 minutes.

6. Pour the supernatant into a clean SS-34 tube through a small two-ply square of cheesecloth placed in the center of a funnel. The cheese cloth catches any fragments of SDS/genomic DNA pellet floating on the surface.

7. Precipitate the nucleic acids by adding 20 ml of isopropanol to the SS-34 tube and ice for 10 minutes. Centrifuge at 10 k × g for 10 minutes.

8. Aspirate off all the isopropanol supernatant. Dissolve the pellet in 5 ml of TE buffer (10 mM Tris-Cl, 1 mM EDTA, pH 7.5). Add 5 ml of 5 M LiCl solution to precipitate RNA. Leave on ice for 10 minutes and centrifuge at 10 k × g for 10 minutes.

9. Pour off the supernatant containing plasmid DNA into a clean SS-34 tube. Add an equal volume of isopropanol (10 ml) and precipitate the nucleic acids on ice for 10 minutes. Centrifuge at 10 k × g for 10 minutes.

10. Aspirate off all the isopropanol supernatant. Dissolve the pellet in 1 ml of TE buffer. Transfer TE solution into a 1.5 ml epindorf tube. Add 15 ul of RNAse A solution (20 mg/ml stock stored at -20 °C), vortex and incubate at 37 °C for 20 to 30 minutes to digest remaining RNA.

11. Precipitate the plasmid DNA with PEG solution (30% polyethylene glycol, 1.6 M NaCl) by adding 0.4 ml and incubating 1hr-overnight on ice. This step discriminates very large plasmid DNA from small nucleic acid fragments as only the larger plasmid DNA precipitate.

12. Spin the PEG solution in the centrifuge at full speed for one minute. Aspirate off the supernatant PEG buffer and dissolve the PEG pellet in 0.4 ml of TE buffer. If it is difficult to resuspend with pipetteman, let it sit at room temperature for 10 minutes and try again.

13. Extract proteins from the plasmid DNA using PCIA (phenol/chloroform/isoamyl alcohol) by adding about 0.3 ml. Vortex vigorously for 30 seconds. Centrifuge at full speed for 5 minutes at room temperature. Note organic PCIA layer will be at the bottom of the tube.

14. Remove upper aqueous layer containing the plasmid DNA carefully avoiding the white precipitated protein layer above the PCIA layer, transferring to a clean 1.5 ml epindorf tube. If you catch any precipitate when removing aqueous layer, add fresh PCIA and repeat.

15. Add 100 ul of 7.5 M ammonium acetate solution and 1 ml of absolute ethanol to precipitate the plasmid DNA, usually on ice for 10 minutes. Centrifuge at full speed for 5 minutes at room temperature.

16. Aspirate off ethanol solution and resuspend or dissolve DNA pellet in 0.3 to 0.5 ml of TE buffer. This is the final stock of PEG pure plasmid DNA which is suitable for DNA sequencing and long term storage.

17. Measure the concentration of the plasmid DNA by diluting stock into water at 1:200 or 5 ml of DNA per 1 ml of water (blank spectrophotometer to water). The absorbance at 260 nm multiplied by ten is the concentration of the DNA in units of mg/ml for a 1 cm pathlength cuvette (i.e. 50 mg/ml/OD 260nm). A 250 ml flask should yield about 0.5mg of DNA

Buffers

GTE solution

stock solution volume final concentration

40% sterile glucose 2.27 ml 50 mM
0.5 M EDTA, pH 8 2.0 ml 10 mM
1M Tris-HCl, pH 8 2.5 ml 25 mM
sterile ddH2O 93.23 ml
Total: 100.0 ml

Use all sterile stock solutions. Store at 4 degrees C.
--------------------------------------------------------------------------
NaOH/SDS Lysis Solution

stock solution volume final concentration

1N NaOH 2.0 ml 0.2 N
10% SDS 1.0 ml 1%
sterile ddH2O 7.0 ml
Total: 10.0 ml
--------------------------------------------------------------------------
5 M Potassium Acetate Solution

stock solution volume

5 M potassium acetate 60 ml
glacial acetic acid 11.5 ml
ddH2O 28.5 ml
Total: 100 ml

Filter sterilize. The resulting solution is 3 M potassium and 5 M acetate and has a pH of about 4.8.


OR use the following recipe is you don't have KOAc

glacial acetic acid 29.5 ml
KOH several pellets
ddH2O to 100 ml
Total 100 ml

Add the KOH pellets until the pH is 4.8.
--------------------------------------------------------------------------
TE Buffer

10 mM Tris-Cl, pH 7.5
1 mM EDTA

Make from 1 M stock of Tris-Cl (pH 7.5) and 500 mM stock of EDTA (pH 8.0).
--------------------------------------------------------------------------
PEG solution

30% PEG 8000 (polyethylene glycol)
1.6 M NaCl

Store at 4°C, but you can get away with storing at room temperature.

Iqbal said:

I am trying to isolate plasmid from wild type bacteria and having problems in purification. First of all do they degenerate more rapidly and secondly are there any protocols for purification from other contaminents like RNA and chromosomal DNA (no kits).

Hello! Since this is a wild type plasmid, and as such may be unstable in the strain you've got when you culture it in lab conditions, you might first want to optimize culture conditions to as closely mimic the conditions where the bug is naturally found. Once you get this step done, the standard Sambrook et al protocol, with EDTA present in all storage buffers, or the large scale protocol that Frasemoss has elaborated, should enable you to get the plasmid DNA you want.

Thanks. This seems to work better.