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Problematic DNA Extraction

extraction pcr dna

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#1 hmwmx

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Posted 14 April 2017 - 08:24 AM

Hello, 

 

Our lab has noticed some green/black contamination in a solution of 30% (w/v) PEG 400, 0.1M Na HEPES (pH 7.5), and 0.2M MgCl hexahydrate which we believe to be bacterial or fungal (or both) and we would like to attempt PCR sequencing in order to identify the offending microorganism.

 

Unfortunately, extracting any DNA (if present) is proving rather difficult. We have attempted mechanical lysis using glass beads, however, that has not yielded any positive results.

 

I think we'll likely spin the contamination down, re-suspend the resulting pellet in PBS and attempt enzymatic lysis, however, I thought I would enquire as to whether anyone have any experience of extracting bacterial/fungal DNA from unusual chemical solutions?

 

If anyone has any literature in mind some direction would be greatly appreciated...

 

Thanks.



#2 merlav

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Posted 26 April 2017 - 06:09 AM

I don't know if you already did the sequencing. As a lab manager myself Im trying to understand what benefits will bring it.  Did your lab have a kit that want to optimize? Other than this I can't find a valid excuse to use so expensive resources/reagents.  A lab is always fighting against environmental contamination the better approach is having good laboratory practice, propers aseptic techniques  and using good cleaning agents.  A cleaner that eliminates spores of surface is less expensive than sequencing and because it usually is very concentrate so it will last for long time. Using autoclaved/baked glassware also helps to keep microorganism away. Prepare the quantity needed, for example if you will use 100-250 mL of a buffer and it isn't use very much then why prepare a 1L? is better to prepare up to the double that you will need. Every day all surface most be cleaned with 10% Lysol (or similar) followed by 70% EtOH, change gloves frequently and when using it keep your hands away of phones, keyboards, face and hair.  Identify possible contamination sources and keep them clean.

 

Getting back to the original question if you most identify the contaminant, then  first you need to know what type is. Is a "hairy" ball? then is a fungus, it looks turbid? then probably is a bacteria or maybe a yeast?  Then choose the best  technique to eliminate the cell wall for each type of organism. I don't recommend the use of glass beads for so fragile sample it will shred also the DNA.  Spin down the organism, decant buffer taking care of not contaminate anything more at the lab, resuspend in a buffer with the proper enzyme to eliminate the wall, incubate, add the lysis buffer (to eliminate the cell and nucleus membrane), incubate, continue with the DNA purification protocol of choice.  It could be column base, salting out or organic extraction (phenol/chloroform).  


Science without religion is lame, religion without science is blind.
Albert Einstein

I am among those who think that science has great beauty. A scientist in his laboratory is not only a technician: he is also a child placed before natural phenomena which impress him like a fairy tale.
Marie Curie





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