Hello fellow researchers. I am struggling to find a good plasmid miniprep protocol for Gram-positive bacteria, specifically Lactococcus lactis. I have followed several protocols on PubMed, and I have also tried to dig through previous questions with little to no luck. One such example is the paper "Rapid Mini-Prep Isolation of High-Quality Plasmid DNA from Lactococcus and Lactobacillus spp." (O'Sullivan and Klaenhammer 1993). The problem I am having, is that I am getting chromosomal DNA in all of the preps that I attempt. I am trying to determine if my plasmid is present, and I have tried several methods such as colony PCR with no success due to negative control contamination due to our building's air quality. Basically, I have been doing variations of lysozyme pretreatments of 20-50 mg/mL at 37 C for various times. I have followed protocols as best as I can, but I keep getting smears of DNA. My gels are done properly, so I know that is not the issue. Can anyone point me in the right direction? Or possibly provide me with a protocol that is more updated than 1993? I appreciate any and all help.
Dear fellow,
You may like list out your problems serially so that one can give inputs/suggestions.However, these few points are appended here which may help you out some extent -
(1) The smearing of DNA bands are the results of presence of DNA degrading agents in your purification reagents/materials. Ensure that all the items you use are free from DNase or similar agents capable of degrading DNA.
(2) Ensure the buffers used are of basic pH value. An acidic pH environment is responsible for the depurination of DNA helix resulting in to breaks in helix (eventually smearing)
(3) Ensure that the percentage of agarose gel is considerable higher enough to separate/resolve the plasmids in the gel otherwise smaller plasmids may run out of the gel during electrophoresis run.
Good luck,
Dr. A. K. Dubey
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