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Autoclaving PCR waste in the room where PCRs are set up and run - Is it a proble

PCR RT-PCR Contamination Waste Management Aerosols

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#1 GrapesMcGee

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Posted 12 June 2014 - 02:38 AM

Hello All, 

 

I work in lab set up last year. The lab is made up of 2 rooms, one for preparing, running and analysing gels with the other room for everything else - which at the moment is mainly preparing and running standard PCR reactions and RT PCR reactions as well as DNA and RNA extractions etc.

 

In this main room is a 60L (medium sized) autoclave, which is mainly used for autoclaving waste tips and tubes used for setting up, loading gels or analysing reactions, it is also used to to kill biological waste from another lab. 

 

The autoclaves has a metal steam exhaust pipe which condenses it to liquid which then is fed into a standard drain poly-pipe, with no obvious seal. The bottom water that remain in the autoclave after a run is regularly changed and the machine cleaned. Some times the autoclave is used for steralising new plastics - but for my purposes I have been ordering sterile plastic consumables. 

 

Plastic ware, tubes etcs are kept loosely covered, in closed bags or boxes within the same lab, normally on the bench tops and shelves. 

 

My question is: In your opinion, would this set up give aerosol contamination of PCR products which are present in the waste? And could this land on surfaces around the lab, or remain in the air, enough to cause a significant contamination problem in newly set up reactions for the same PCR product?

 

I have only found 1 paper that has addressed this issue precisely - http://www.biotechni...ues-348850.html

Which was quite useful, but of course still leaves me questioning smile.png.  

 

In previous labs I have worked in, autoclaving of PCR waste was separated with dedicated autoclave, or other disposal method such as incineration. 

 

The background to my work, is that I am screening a large amount (approx 3000) of different varieties of the same thing for the presence or absence of a genetic marker. Negative controls (NTC) have contamination and it appears the frequency of positives have changed significantly - therefore suggesting false positives - but I can't be sure. I have changed everything, and have been working on this problems for a long time. I've read a lot of the previous discussions on this forum and elsewhere, and although I know that there is possibility of multitude different problems - I feel I have worked through all possibilities. It really is a long story that I can't explain fully on here smile.png.

 

I am just curious for people's opinion about the autoclave issue and waste management of PCR waste? 

 

Many Thanks in anticipation,

 

Grapes McGee


Edited by GrapesMcGee, 12 June 2014 - 02:42 AM.


#2 bob1

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Posted 12 June 2014 - 01:10 PM

The short answer would be yes - pretty much anything that can cause aerosols (including things like opening eppendorf tubes) could function as a source of contaminants.

 

Autoclaving PCR products is useless - the DNA will survive the autoclaving process fairly effectively, the waste would be better off just being sent for incineration.  However, depending on the success of the autoclave at containing any evaporated water and how the door opening is managed (only when completely cool?) then it may not be the cause of your problems. 

 

Is there any chance you could get entirely new reagents, tips, etc, and move to a different room in the same institution to see if that improves your PCR?



#3 Ameya P

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Posted 12 June 2014 - 10:32 PM

If you are quite certain that your false positives are on the rise, you are better off changing the place you use for PCR setups. 

 

In my experience, I have set up PCRs in the same room as the thermal cycler, with out any contamination issues. All you need to do is use a separate table and wipe it with ethanol before and after your work. 

 

Since the lab is new and made up of two rooms, it is unlikely that you can get the autoclave to move. So, just move your setups away from anything you think is affecting the quality of your work, 

 

Ameya


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