I have a lot of experience with extracting BAC DNA. BACs are single copy plasmids with an average insert size of 100 kb, you do not get much tougher than that. We have found that yields are around 25-100 ng per ml of overnight culture, so you can't expect miracles.

We use cesium gradient purification for large scale preps (400 ml), but standard alkali lysis gives similar yields of BAC, but with contaminating chromosomal DNA. The key to getting this to work is to go fast after you have lysed with P2. P1+RNaseA (10 ug/ml), P2 fresh and P3 chilled (400 ul of each per 5 ml of overnight culture in L broth).  Spin down the bacteria and resuspend the pellet in P1+RNase - 10 minutes at room temperature. Add P2, and mix gently until homogeneous. Add chilled P3 and break up the white precipitate to "snowflakes" with gentle mixing.  Spin and recover most of the supernatant, add 0.7 volumes of room temperature isopropanol and spin immediately at room temperature to recover the DNA. Wash pellet in 70% ethanol and resuspend in TE. DNA dissolves quickly so don't worry about insoluble material just spin it out, recover the supernatant and move on. To get cleaner DNA and to concentrate it, re-precipitate with 0.7 vol isopropanol and 0.1 vol of 3M NaAcetate pH5.2. Check concentration on a gel. You will probably be best to extract from 50 ml of culture.

You could try:

http://www.qiagen.com/be/resources/technologies/plasmid-resource-center/growth%20of%20bacterial%20cultures/#Chloramphenicol amplification

Also I've seen  that growing bacteria on agar plates works much

better than liquid culture for problematic plasmids. 

Grow 5ml over night culture. spin down, folllow qiagen protocol, but adjust to  300 ul p1, 300 ul p2, and 420 p3. Elute by adding 50 ul H2O, incubate in 70C for 30 min. Spin down. You will got what you need. Miniprep big plasmids, I always do this way. Yield increase 50 times. 

go old school:

Use sds lysis method on a scaled up culture and purify using CsCl gradient.

option 2, perform an in gel lysis (aka gel plug) and perform a PFGE, elute band from gel.

I gather you are trying to perform a shotgun sequencing on the (mega)plasmid ?

but if you just want to check for insert/select genes you can perform a primer walk sequencing strategy.

I would also go old school and do the standard P1, P2, P3 extraction but with some slight modifications.  Pellet 1 mL of overnight culture, pull off super, add another ml, and repeat the pelleting process 3 or 4 times to get a larger pellet, and then proceed with the extraction.  Alternatively, you can perform 12 1mL preps at the same time from the same overnight culture, do the extractions all the way to the finish, then add 50uL to the first tube...resuspend the pellet and then transfer the entire volume to the next tube and repeat the process.  This will effectively increase the concentration of your plasmid without increasing volume.  

The nanodrop measurement problem is likely to be due to residual chromosomal DNA. This will show up as a high molecular weight band or smear at the top of the gel in uncut DNA, or as a very faint pattern of bands in digested DNA - if your gel is good enough. 

Alkaline lysis methods are not all that good for large, low-copy plasmids, as they rely on the 'snapback' properties of supercoiled covalent closed circular plasmid DNA. As the strands are linked, they cannot drift away from each other after alkaline denaturation and immediately renature after neutralisation. Big plasmids tend to get sheared and the strands separate and are salt-precipitated with the chromosomal DNA and cell debris upon renaturation. 

You need quite a big culture to get large amounts of low copy plasmids - I would recommend about 250 ml. Unless you're equipped to handle large bacterial cultures, this can be quite a challenge: how to incubate with sufficient aeration; how to pellet the cells, among other things. As Barry say's - go to the old school. The old-fashioned Triton lysis method can be recommended. 

I had some success with a rather obscure method called hydroxyapatite column chromotography. You mix your cleared lysate with urea and run it over a bed of hydroxylapatite in a cylindrical funnel fitted with a fritted glass filter. Then you wash the bed with urea solution and elute in a low-salt buffer. It's a bit like using the minicolumn methods, but scaled up 100x. The advantage is that there is essentially no limit to the amount of lysate you can handle. I had a look for the reference, but no luck. Try searching 'hydroxylapatite' and 'plasmid' if you want to try it. The reference will be about thirty years old. 

Thanks everyone. I did try I the old school method i.e. the alkaline lysis method. I have tried different culture volumes, from 50ml to 400ml. Every single time, I get a big white pellet after isopropanol precipitation, followed 70%EtOH. And this pellet NEVER dissolves in TE. I have kept the sample in 65°C and still the pellet never goes into the solution. I am not sure what this white precipitate is? Should I be doing a chloroform step before isopropanol treatment?

@Andrew, I will look into the hydroxylapatite protocol. Thanks  

The Qiagen miniprep kit has always given me great results for plasmid extractions from E. coli.

Hi Preetam, you could try to grow your E. coli in terrific broth (TB) instead of LB, the cultures will grow to much higher densities and typically plasmid yields are a lot higher. Works well for both kit based and old school preps.

Qiagen has several kits for plasmid extraction, each kit's column has different cut-off limit on the plasmid size it can hold. The mini prep kit is not recommended for purifying 40Kb plasmid, especially when it's low copy number. I have tried different kits from Qiagen with no luck.   

@Dirk, thanks for the tip, I will try using TB

If you just want to sequence it you could try templiphi

I have a lot of experience with extracting BAC DNA. BACs are single copy plasmids with an average insert size of 100 kb, you do not get much tougher than that. We have found that yields are around 25-100 ng per ml of overnight culture, so you can't expect miracles.

We use cesium gradient purification for large scale preps (400 ml), but standard alkali lysis gives similar yields of BAC, but with contaminating chromosomal DNA. The key to getting this to work is to go fast after you have lysed with P2. P1+RNaseA (10 ug/ml), P2 fresh and P3 chilled (400 ul of each per 5 ml of overnight culture in L broth).  Spin down the bacteria and resuspend the pellet in P1+RNase - 10 minutes at room temperature. Add P2, and mix gently until homogeneous. Add chilled P3 and break up the white precipitate to "snowflakes" with gentle mixing.  Spin and recover most of the supernatant, add 0.7 volumes of room temperature isopropanol and spin immediately at room temperature to recover the DNA. Wash pellet in 70% ethanol and resuspend in TE. DNA dissolves quickly so don't worry about insoluble material just spin it out, recover the supernatant and move on. To get cleaner DNA and to concentrate it, re-precipitate with 0.7 vol isopropanol and 0.1 vol of 3M NaAcetate pH5.2. Check concentration on a gel. You will probably be best to extract from 50 ml of culture.

Dear friend i am also working with some low copy number plasmids. but isolating plasmids through kit are better for sequencing. In my lab we are using Hoefer fluorimeter for the estimation of DNA concentrations by using hoechest dye. Nanodrop doesn't show the exact reading. I cant relay upon nano drop. But the readings from the fluorimer is reproducible and is accurate also. For better yeild i isolate the low copy number plasmid by manual Alkaline lysis method only. after adding Na acetate we spin the sample and the top liquid phase was aliquated into many 1.5 ml tubes. after 1st precipitation step ae add TES buffer 200 micro lt. after dissolving pellete we pool 3-4 sample together so that we could concentrate the plasmid and proceed for further steps. this method gives good yield of plasmids. Sigma kit also gives good concentration of plasmid DNA.

All the best.

Try colony PCR and sequencing the PCR product. I do this to sequence E. coli chromosomal DNA all the time (using Takara/Clontech SapphireAmp... super cheap and much better than anything else I've tried). I've amplified and sequenced up to ~6 kb chromosomal DNA this way, occasionally using touchdown cycling to avoid non-specific products. Of course, if you have to purify the plasmid anyway for some downstream step you should do the midi prep.

I often had to amplify the DNA region by PCR to obtain a reasonable yield.

That was the only way to sequence many difficult clones. Just make sure to pick the correct primers. I have tried using different DNA extraction columns but that was not the solution. 

Good luck

The problem is the low concentration of plasmid DNA re all the rest of the bacterial extract. One way to increase yield is to block protein synthesis of the bacteria (this does not stop replication, but does de-repress the colE1 origin).

1.  Morning : Inoculate 400 ml culture medium (with appropriate antibiotic) with 8 ml of bacterial pre culture (small overnight culture).

2. Incubate with vigorous shaking at 37º C until the OD600 is approximately 0.4.

3.Add chloramphenicol to a final concentration of 170ug/ml.

4.Incubate for a further 12 to 16 hours (overnight for example) at 37º C with vigorous shaking.

Yes, the suggestion of Ian is better. I am using this several times. You do not need 400 ml culture, sometime 5 ml is enough and use 170ug/ml chloramphenicol. Adding chloramphenicol is a good idea for any kinds of copy number.

Below is some links for ref: 

http://www.nhm.ac.uk/content/dam/nhmwww/our-science/dpts-facilities-staff/Coreresearchlabs/chloramphenicol.pdf

https://www.ncbi.nlm.nih.gov/pubmed/3545722

https://www.ncbi.nlm.nih.gov/pubmed/3545722

The question was three years ago!

Preetam Janakirama Hi

I came across this question today, as Im facing the same problem. I have a low copy number large plasmid in E.coli that I am unable to isolate.

Could you advice me on what you did finally to successfully isolate the plasmid ?