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Plasmid isolation from Enterococcus


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#1 2xzwei

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Posted 06 September 2013 - 04:00 AM

Hi all!

Is there anybody out there who has experience concerning plasmid isolation from Enterococcus (or gram-positive)?

I tried it for weeks now but I cant get it work.

 

Initially I tried simple plasmid prep with a kit (Qiagen), but no plasmid yield at all.

So I found out that incubation with Lysozyme and Mutanolysin could help.

I tried different concentrations (1mg/mL up to 30mg/mL) and different incubation times (15min to several hours) but I never could isolate a plasmid. (Lysozyme stock prepared in 10mM Tris-Buffer pH 7.5).

 

Curiously I always had good DNA concentration and nice peaks (250ng/µL or more - using Nanodrop technique).I loaded abundant of the samples on a 1% gel but I just god very faint bands at around 10kB (looks like I just loaded 10ng of DNA). Also restriction digest and PCR doesn't work - no cleaved products, no amplified bands by specific primers that work for the plasmid in E. Coli.

 

My last resort now was to try heat lysis...initially I started with 95°C for 15 minutes and now expanded to 30minutes at 105°C. I also tried to heat-lyse in STET buffer (containing EDTA, Triton-X, Tris and Sucrose at pH 8.0) but it doesn't work (even preincubation with Lysozyme didn't help).

 

So...I'm totally clueless at the moment what is wrong. The bacteria definitely must contain the plasmid (9kB) as I electroporated them and they are growing fine on Erythromycin plates (100µg/mL).

 

Some hints, tipps, workarounds would be appreciated...

 

Thank you!

 

pmspverdau_nachlysozym.jpg



#2 bob1

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Posted 07 September 2013 - 02:16 PM

I don't have any tips for extraction from gram + bacteria, but there are a few threads on here about it.

 

The DNA quantity vs not seeing it on the gel probably indicates that the DNA is very degraded (this will still show up as DNA on a nanodrop or any other spec) or that you are extracting RNA as well.






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