The microbes which their DNA is to be extracted,where incubated in broth for 15hrs before transported. Out of 32 isolates only 11 bands were seen when the DNA was viewed on gel.
Dear Frances
There could be numerous reasons for not getting band in the agarose gel after DNA extraction. Can you be more explicit regarding your extraction procedure like which protocol you have followed, and it would be much better if you can share your gel image.
Dear frances
There are many reasons for not getting DNA band. May be number of bacterial you took for isolation is not sufficient, are else u need to see the DNA extraction protocol once again.
Did you use a lysostaphin/lysozyme combo or lysozyme pre-treatement before extraction. Some bacteria are restistant to the lysis buffer that you use. Treating the bacteria with cell-wall degrading enzymes weakens their cell wall and helps increase lysis efficiency.
Maybe you should show the gel and explain what you do, extraction method and the organisms you are working with, including the reagents with what you are running your gel.
I guess you used different microbes suggesting that you should have used different conditions for extraction.
This is my understanding:
You had 32 isolates. You grew them 32 isolates individually, and extracted the genomic DNA from all of them. Then you ran the isolated gDNA on a gel. You supposed to see 32 bands from 32 samples, but you only observed 11 isolates with a band.
Except extraction problems mentioned above, could it also be due to the other 21 isolated did not grow well in the same specific medium you used? Did you check OD600 for each growth before you used them for gDNA isolation? Or at least observed some degreed of 'turbidity' in the cultures? Or did some of the isolates had died??
Have you checked amongst the 32 bacterial strain, which is Gram positive and Gram negative. Normally, you would need lysozyme or lysozyme/lysostaphin combo treatment prior to lysis.
From what I see on the gel, the DNA is getting degraded. This may be one of the reasons there was no DNA in the other wells. Do the following the next time time you do DNA extraction to avoid DNA degradation:
1. Perform Extractions at 4°C, on ice or in the cold.
2. Inhibit nuclease activity.
3. Store purified DNA correctly.
All the best.
It did look like that the DNA quality is not good:
1. Did you use a commercial kit or home-made solutions to isolate the genomic DNA?
2. Did you check the 'purity' of the gDNA? Check the OD260/OD280 ratio for an indication of nucleic acid purity. Pure DNA has an OD260/OD280 ratio of ~1.8. [ http://cshprotocols.cshlp.org/content/2007/11/pdb.ip47.full ]
3. It will be good if you can answer some of the questions people posted, so people can properly diagnose the problems.
Pay attention : gram positive and gram negative strains do not possess the same extraction rate. There are specific protocols for each.
you should seperate gram negatives from gram positives
It will work for you :)
I use this protocol for extraction (Marmur 1994).
May be you extracted DNA by boiling method in this method is weak band, but this amount of DNA is enough for PCR. DND extraction method mentioned in the our article is appropriate for PCR.
Article O-serotyping of Escherichia coli Strains isolated from Patie...
One of the reasons could be that the growth rates of different isolated may differ under the conditions used which is reflected in the DNA extraction. You may try another prolonged incubation period as well
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