Low copy number plasmids

[Hercules Rockefeller]

What are people’s tips and tricks for maximising the yield of low copy number plasmids in maxi/midi preps? I’m dealing with a low copy number plasmid that I simply cannot prep.

Here is the stuff I know already….

-- Increased culture volumes

-- Chloramphenicol amplification of plasmids

-- Use Terrific Broth instead of LB


These haven’t had much of an impact so far. Are there specific strains of E.coli that are better for low copy number plasmids?

Cheers.

 

[S.A.M.]

How large is the plasmid? Can you reduce the volume of your prep?

 

[CharonZ]

Which kit are you using (if at all), is it silica based?
Do you get nothing at all? If so, are your growing under selective conditions? Are you sure that the plasmid is in the cell and e.g did not get integrated into the genome or otherwise lost (stupid question, I know, but it happens...).

Is the used E. coli strain res and end negative (these significantly reduce yield)?
Do you have an estimate of expected copy number?

Some other suggestions depend on the kits you used (or are you doing manual alkaline lysis)?

 

[Hercules Rockefeller]

Which kit are you using (if at all), is it silica based?

Qiagen maxi/midi preps which are, AFAIK, silica based columns.

Do you get nothing at all? If so, are your growing under selective conditions?

I’m getting very low yields of plasmid. The plasmid is commercially purchased with neoR and ampR markers. Cells have been grown with amp (100ug/ml).

Is the used E. coli strain res and end negative (these significantly reduce yield)?

I’m using TOP10 cells from Invitrogen.

What is “res”? Did you mean to say “rec”? TOP10 cells are endA1 and recA1 which indicates (I think) that they are endA1 and recA1 negative (?). Being negative for rec and end is a good thing (AFAIK) as this helps to prevent recombination and rearrangements between repeated sequences. The plasmid does contain some repeated motifs.

Do you have an estimate of expected copy number?

Actually, I’m not sure as to the expected copy number.

How large is the plasmid? Can you reduce the volume of your prep?

7.1 kb.

I could reduce the volume, but why would I want to?

 

[S.A.M.]

I’m getting very low yields of plasmid. The plasmid is commercially purchased with neoR and ampR markers. Cells have been grown with amp (100ug/ml).

7.1 kb.

I could reduce the volume, but why would I want to?

With the Qiagen maxiprep, if you reduce the final volume by half [50 microlitres] and also reduce the volume of the prep [try 6 ul], it helps to give better yield and more distinct bands. Large plasmids tend to a low copy number. What is yours? 4-5? Or even less?

Also depending on what you are harvesting it from, I have heard you can amplify with chloramphenicol before harvesting, but I haven't tried it myself.

When low-copy-number plasmids containing the pMB1 or ColE1 origin of replication are prepared using QIAGEN Plasmid Purification Kits, plasmid DNA yields can be improved by adding chloramphenicol to the culture medium (170 mg/liter) to amplify copy numbers. For details and precautions on the use of chloramphenicol when culturing plasmids, please refer to standard manuals for cloning procedures (e.g., Molecular cloning : A laboratory manual / T. Maniatis, E.F. Fritsch, J. Sambrook).

Cultures of bacteria containing low-copy-number plasmids amplified in the presence of chloramphenicol should be treated as if they contain high-copy-number plasmids when choosing the appropriate culture volumes for the QIAGEN-tip to be used.

http://www1.qiagen.com/FAQ/faqview.aspx?faqid=3&ShowAll=1&FaqCategoryId=0&MenuItemId=56

Even spectinomycin has been used for amplification, but I don't have a concentration.

Most of the time I just call the technical support and they give you some good ideas.


edit:Forgot one very simple thing. Have you tried letting the columns elute in a cold room overnight? I hear that also increases the yield dramatically. [word of mouth]

 

[CharonZ]

Hmm, I think with Qiagen I always had a lower yields with low-copy plasmids than with Macherey Nagel kits. But then in my case the size might have also been a problem (I had 100kb plasmids). With below 10 kb plasmids I did not have that much of a trouble using Qiagen. The yield were lower of course, but sufficient for my needs. I assume you have followed the modifications given in the manual (PB-buffer and maybe pre-warming the elution buffer?).
Just for comparison purposes using the Qiagen Maxi kit for low copy plasmids (around 1 copy per cell) in Gram- positive bacteria I got around 5 microgram per gram wet weight.

I used res- too loosely. I meant whether it contained any restriction systems (like mcr, though in your case it probably is not a factor). And I also meant recA negative, but forgot to mention it. Sorry about that.

But Top10 should fit the bill in these regards. I recall that sometimes the lysis is not as thorough as with other strains, especially if the volume is increased (it is apparently also true with Top10F'). I think it was even worse if you grew them in TB. I always assumed that it was due to additional sugar production. It may help to increase the volume of the lysis buffers relative to the culture volume. Other than you could try other kits. As already mentioned I had good experience with Macherey-Nagel kits, but there are also specialized low-copy plasmid kits. I have not used them, though.

 

[Hercules Rockefeller]

Okay, thanks S.A.M. & CharonZ. There are a couple of different ideas and suggestions in there. I'll do some tinkering and see how I go.

 

[CharonZ]

Well, the most obvious one is chloramphenicol amplification, but Hercules already did that. Elution in a cold room is pretty much a myth. At lower temps DNA sticks even tighter to silica. Slow elution sometimes helps, though, but this is temp independent. Or rather, for plasmids bigger than 10 kb a higher temp does help.

 

[S.A.M.]

If nothing else works, I go back to good ole alkaline lysis. But I use E. coli not sure about your cell line.

 

[CharonZ]

Hercules said he uses Top10, which (if memory serves), has all the nice and good deletions.

 

[S.A.M.]

I meant I haven't used it. :shrug:

Most of my work deals with environmental bacteria [Bifido, Bacteroides, Lacto].


Right now, I am trying out Sigma's GenElute Plasmid Midiprep. It looks pretty straightforward.

http://www.sigmaaldrich.com/sigma/general information/pld35inf.pdf

 

[CharonZ]

Well, most environmental bacteria are tougher (mostly due to higher cell stability). Enterobacteria in general are still pretty easy (unless you got them in a nasty biofilm), but soil bacteria or certain Gram+ bacteria are nasty as hell. Usually one has to do use some nastier lysis methods, but then the chromosome integrity is a problem. Fortunately this is less of a prob for smaller (less than 100-50 kb) plasmids.
By comparison E. coli are wimps. Which is one of the reasons why they are used as a basic work horse.

 

[S.A.M.]

I have poo samples, plenty of DNA. I'm trying out this kit because the last person who used it got a pretty gutenkleen yield. Doing it on Monday, lets see how it goes.