DNA contamination during concentration using a speedvac

[BabcockHall]

On a discussion board about DNA sequencing I found this comment:

"Instead of evaporating all of this, we bind it all to AMPure beads (2x cut), wash the beads once in 70% ethanol, dry them, and resuspend them in 10.5ul of hyb buffer. It takes all of 5 minutes, and no cross-contamination prone speedvac step!"

 

I also found this comment elsewhere: "Particular care should be taken to avoid contamination of commonly used rotors. " From the context of the article, the author may have been referring to rotors used in organic precipitation of DNA or in post-precipitation drying of the pellet in a speed vac, but I am not sure. I also found ThermoScientific's literature about one low vacuum pump that was claimed to be hermetically sealed to prevent contamination.

 

I am interested in the use of PCR in forensics. How much of a problem is contamination from other DNA during concentration in a speed vac? Is it the rotor that is the biggest problem, or is it aerosol DNA? How do people avoid it? Thank you.

[DrP]

I do not know for sure as I am not a biologist, but have used rotovaps before... Wouldn't a thorough soak and scrub in an acid bath, followed by a base bath and multiple rinses be enough to get rid of any trace DNA contamination in the rotovap flasks? Or, if you have the budget for it, just use a new flask for each test.... it depends on who is paying for the tests to be done and to what level of purity they want to work with. They may consider paying an extra £30 per run to ensure there is no cross contamination by eliminating the possibility by using completely new glassware. ??

 

(***£30 was just an estimate - I'm not sure how much new flasks cost for rotovap kits.***)

[BabcockHall]

Hi DrP, Bleach solutions are particularly effective at removing DNA contamination, more so than acid (Prinz and Andrus, Biotechniques; see also the Promega guidelines). A speedvac is a slightly different beast than a rotary evaporator, but I do see some similarities. In both cases, releasing the vacuum seems as if it might be a crucial step. If there were airborne DNA, it could sneak in at that point.

[CharonY]

Depends, really. Most modern speedvacs are pretty good in timing the onset of centrifugation (if you do it manually, some people switch it on too early) and have a vent that allows slow release while still centrifuging down.

Thus, even if you load everything up chances are that you will not observe cross-contamination. However, general contamination can get into the sample fairly easily if the area is prone to any kind of contamination as the samples remain open for an extended amount of time.

If everything is kept clean, contamination is rarely an issue (I have run speedvacced samples through MS or did sensitive PCRs with no indication of contamination of any kind, for example). However, in shared labs where the instrument is used (and abused) for all kind of stuff, chances rise dramatically that the area and the instrument itself gets dirty.

 

So unless you have juryrigged system of dubious origin, the main issue is tends to be cleanliness and not so much the process or instrument itself (cleaning out rotor and chamber is mandatory).

Edited March 10, 2015 by CharonY

[BabcockHall]

I may not be picturing this accurately, but what about the air that comes in when one releases the vacuum? I would think that some kind of filter is necessary.

[CharonY]

Not necessarily. For critical applications you would place it in a clean area, anyway. But often you have equal or larger risks on the way, unless you work in a zero flow box. In that case it helps if you place it under a horizontal clean bench. Sample-to-sample contamination typically does not happen (or at least I found no evidence for it when I evaluated the system).

 

That being said, I am not aware of speedvacs with HEPA filters though some freeze-drying systems have them (but the vacc is typically higher there, too, resulting in more turbulences).

Edited March 11, 2015 by CharonY