Problems with plasmid isolation

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Problems with plasmid isolation

Postby DFRevell7 » May 29 2007 10:53 am

Hi

We have been trying to isolate plasmid DNA from minipreps grown during the day and/or overnight in LB + 100ug/ml ampicillin. The vector is pGEX based (pGEX4T2 and pGEX6P1) and come from Amersham Biosciences. Using kits from Sigma or Qiagen both result in poor yields of plasmid irrespective of the incubation period. Despite the fact that these vectors do have the pBR322 origin of replication and so give lower yields, never encountered this before. Also, have just bought in a cloning vector - pSP72 - which has the pUC origin of replication and even with this, we get low yields - 35 to 50ng/ul. Have checked the kits, the media, the selection and the strain ( we use E.coli TOP10 or DH5alpha).

We are now trying to amplify our plasmid yields using spectinomycin in order to try to get better yields of plasmid. never encountered this problem before but would welcome any tips/suggestions. thanks.


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Postby relaxin » May 29 2007 11:15 am

Sometimes the yield of a plasmid depends on the insert sequence. I have experience with low yield of some luciferase reporter constructs, but not others.
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Postby DFRevell7 » May 30 2007 3:12 am

Sure.......did wonder if the gene at the 3' end of the GST tag is toxic to the host. However, the gene of interest has been generated using oligos with a high E.coli codon bias. It could be that we are still getting basal expression and that the protein is still exerting a toxic effect. However, it doesnt account for why a plasmid like pSP72 gives low yields too as a control plasmid ?!

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Postby feyenoord forever » May 30 2007 4:35 am

Maybe do a new transformation, pick single colony and so on; also your ampicillin concentration is high, try 50 µg/ml.

You can put the plasmid you isolated on gel, see how it looks; even do a restriction analysis
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Postby DFRevell7 » May 30 2007 4:50 am

Ok.....but use 100ug/ml ampicillin in our cultures routinely without problems.
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Postby feyenoord forever » May 30 2007 6:39 am

DFRevell7 wrote:Ok.....but use 100ug/ml ampicillin in our cultures routinely without problems.


99 out of 100 times it's no problem but here I also had the same problem with a lentiviral plasmid and reducing the amp concentration helped in my case.

You can also try rich medium (like TB, SB) but be aware that you don't overgrow your bugs, they'll reward you with low yields
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Postby DFRevell7 » May 30 2007 10:20 am

One thing I suspect is that these pGEX vectors are a bit 'leaky' and if the host doesnt like the protein being expressed, even at a basal level, then yields of plasmid can be lower. I have experienced this with pET vectors especially - some proteins are just very toxic and I found I had to transform the plasmid into fresh cells each time I wanted to express them to obtain protein !!

I will try different amounts of ampicillin just to see though.

thanks


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Postby Dr Rich » Jun 01 2007 4:34 am

Have you run an analytical gel on your plasmid preps? Save samples of lysate and all flow throughs from the column preps, precipitate the DNA using etoh and run on a gel (methods for this in the back of the Qiagen handbook). This will give you a good indication of where the plasmid is being lost. e.g it's either not replicating or it is being lost from the column prior to elution. You should definitely start with a fresh streaked plate, make a pre-culture and use that to inoculate your main culture. Also with top10 cells these grow v quickly so it is wise to check the number of cells that you are preparing to make sure you aren't overloading the columns. Also do your plasmids have pMB1 or ColE1 origins of replication? If so, for low copy plasmids you can often boost plasmid production by adding chloramphenicol at 170mg/l
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Postby DFRevell7 » Jun 01 2007 4:48 am

We have tried spectinomycin which behaves the same way as chloramphenicol and did improve yields, though that was for a 100ml maxiprep grown during the day until OD @ 600nm was 1.0, adding the spectinomycin to 100ug/ml then leaving to grow overnight.
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