DNA Sequencing Problem - (Oct/09/2008 )
Hi,
I sent my plasmid (purified from E.coli BL21) to a company for sequencing the insert. I got an Info, that sequencing was not possible because my sample was not clean enough (had RNA and Protein within). I used the High Pure plasmid isolation kit from Roche. For plasmid elution I used aqua dest.
Do you have any idea, where the problem might be and how to improve plasmid purification?
How important is the dna concentration? I measured it with extinction at 260 nm.
Thank you very much for ideas! 
I sent my plasmid (purified from E.coli BL21) to a company for sequencing the insert. I got an Info, that sequencing was not possible because my sample was not clean enough (had RNA and Protein within). I used the High Pure plasmid isolation kit from Roche. For plasmid elution I used aqua dest.
Do you have any idea, where the problem might be and how to improve plasmid purification?
How important is the dna concentration? I measured it with extinction at 260 nm.
Thank you very much for ideas!
This could be due to the tecnique in operating or using the kit, i do trust product from Roche but in any case, Qiagen is probably the best in term of DNA quality. Please be more careful in doing the extraction. and check for 260:280 ratio and also 260:230 ratio. elute DNA in H20 ( miliQ ) for best result.
RNA contamination = Rnase gone bad
Protein contamination = u might have accidentally get some white pellet into the column.
Tell me how you extract the plasmid in detail. maybe we could troubleshoot from there.
Preparing plasmids from BL21 strains is a bad idea. The strain is endA+, and will degrade DNA easily during preparation. Reclone the plasmid in a cloning strain such as DH5a, DH10B, Top10, XL-1 Blue or some other endA- strain.
You could try the plasmid purification kit from Qiagen too .
THey claim that this Buffer PB can remove residual endonuclease activity.
For more information kindly read this
http://www1.qiagen.com/literature/brochure...df/seqguid2.pdf
they did mention something on working with endA+ strain and sequencing
@phage 434
Thank you for your hint! Unfortunately we only have the BL21(DE3) clone and not the cloning/ligation products of vector+insert. So is it possible to isolate the plasmid from these cells without damage and to transform them into other cells like BL21 Gold (which are endA-), we also have in the lab? I mean how can I isolate this plasmid without any damage? I will now try the kit from Qiagen.
The plasmid is in BL 21 cells because we need strong overexpression of the protein, so that is why we don't have it in "cloning cells". But now I want to do Site directed mutagenesis on the gene. So do you think I should transform the plasmid to the BL21 (DE3) Gold strain and after that check through sequencing if the insert is ok and then start mutating it from that bacterial host?
@Hamming86
Thank you very much! I will try the Qiagen Miniprep Kit and I will report you the results! I will also have a better look at the white pellet to avoid protein contamination.
Thank you both for more answers! 
Sometimes another purification step helps clean up the DNA. For example, after your miniprep, perform a PEG precipitation or an extra isopropanol/ethanol precipitation. You will lose some DNA so start with a good amount of culture. Overloading the column with too much culture can also produce dirty DNA because there are too many contaminants in the preparation for the column to filter them out and produce clean DNA. So use just the recommended amount of culture. Some strains are also more prone to producing poor DNA for sequencing than others for some reason, so changing to a different strain can make a difference too.
I would transform your plasmid isolated from BL21 into a strain such as DH5a, then miniprep the plasmid from the DH5a strain. The transformation should be very easy with either chemical transformation or electroporation.