I have a wild type bacteria culture and want to check the presence of plasmids in it. I followed the normal plasmid isolation procedure and got a small pellet. In gel electrophoresis a lower intensity band also visualized. The problem is its size is similar to the genomic DNA when they run parallay on the same gel. Gel was 0.7% agarose gel in 0.5x TBE buffer. I did restriction digestion with Not1, BamH1, Hind111. but could not resolve visualize clear bands on the gel.
Standard alkaline lysis methods do not work well with large plasmids. This is because they rely on the plasmid remaining in the supercoiled covalently closed circular form and rapidly renaturing on neutralisation. Large plasmids are easily sheared and are poorly enriched. In addition, they often have sizes well in excess of 40 kb, which is the normal size for chromosomal DNA fragments sheared by pipetting.
The best methods for screening for large plasmids are those that involve a minimum of manipulations. One old method involves applying a colony directly into the well of a gel, adding SDS and mixing with a toothpick. The chromosome remains more-or-less intact and is too large to enter the gel, but plasmids can. Unfortunately, restriction digestion is out of the question and you are likely to get three bands (linear, open-circular and closed-circular) and if there is more than one plasmid, your pattern can be quite complex. Do perform replicates, and if you really want to be certain which band is which, cut them out, elute them and do a restriction digest. In situ digestion is also an option.
Note that standard pipette tips are a killer for large plasmid preps, because they shear all DNA to a size of about 40 kb. People who work with large plasmids often cut off the ends of their pipette tips to make the orifice larger, which increases the shear size. Vortexing is another thing to avoid as it also generates powerful shear forces.
I think that your plasmid DNA is contaminated with your genomic DNA. I suggest that you redo the procedure for plasmid extraction and be careful at the alkaline lysis step as this is very critical. I also suggest that you increase the agarose concentration to 1% for better separation.
Plasmids will give you discrete bands on the gel while genomic DNA will give a smear upon restriction digestion.
If you are getting smears, then most probably its the genomic DNA.
thanks all', actualy i do not know the exact size of plasmids as it a unknown wild type sample.and i got a smear for g DNA digestion but not in suspected plasmid sample.but m
Standard alkaline lysis methods do not work well with large plasmids. This is because they rely on the plasmid remaining in the supercoiled covalently closed circular form and rapidly renaturing on neutralisation. Large plasmids are easily sheared and are poorly enriched. In addition, they often have sizes well in excess of 40 kb, which is the normal size for chromosomal DNA fragments sheared by pipetting.
The best methods for screening for large plasmids are those that involve a minimum of manipulations. One old method involves applying a colony directly into the well of a gel, adding SDS and mixing with a toothpick. The chromosome remains more-or-less intact and is too large to enter the gel, but plasmids can. Unfortunately, restriction digestion is out of the question and you are likely to get three bands (linear, open-circular and closed-circular) and if there is more than one plasmid, your pattern can be quite complex. Do perform replicates, and if you really want to be certain which band is which, cut them out, elute them and do a restriction digest. In situ digestion is also an option.
Note that standard pipette tips are a killer for large plasmid preps, because they shear all DNA to a size of about 40 kb. People who work with large plasmids often cut off the ends of their pipette tips to make the orifice larger, which increases the shear size. Vortexing is another thing to avoid as it also generates powerful shear forces.
I voted that Andrew Jenkins has an interesting answer that rings true with my knowledge.
Years ago we relied on a method published by Clarence Kado (Kado, C. I., and S.-T. Liu. 1981. Rapid procedure for detection and isolation of large and small plasmids. J. Bacteriol. 145:1365-1373.). It does a good job of eliminating most of the chromosomal DNA and then the large uncut plasmids are generally seen quite clearly on ~0.7% agarose TBE gel.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques