I am reading up on preparatory work on working with nucleic acids and a lot of the instructions speak on excessive procedures on cleaning environments with high %ethanol and making sure the equipment is nuclease free, and autoclaved.

Are these sterilization steps really necessary when doing research /running gels? Buying all of this equipment seems very excessive, and given that just one nuclease could compromise my results why bother?

  • 4
    $\begingroup$ If in doubt, add controls. $\endgroup$ – Superbest May 24 '15 at 0:26

Short answer: Yes

Long answer: Depends what you are working with.

DNA: If you are working with DNA, its pretty stable and you can usually get away with a 70% ethanol wash/autoclave (mainly to prevent contamination and obtain consistent results). EDIT: Read Chris's answer also below

RNA: If you are working with RNA well.. whatever you did for DNA doesnt apply anymore. You have to take it to the next level. You will need to replace everything, you'll need nuclease free water, tubes, reagents and whatnots. Throw out ethanol and bring in RNAZap, DEPC water or something like that.

An RNase free environment is essential when working with RNA samples. There are two main reasons for RNA degradation during RNA analysis.

First, RNA by its very structure is inherently weaker than DNA. RNA is made up of ribose units, which have a highly reactive hydroxyl group on C2 that takes part in RNA-mediated enzymatic events. This makes RNA more chemically labile than DNA. RNA is also more prone to heat degradation than DNA.

Secondly, enzymes that degrade RNA, ribonucleases (RNases) are so ubiquitous and hardy; removing them often proves to be nearly impossible. For example, autoclaving a solution containing bacteria will destroy the bacterial cells but not the RNases released from the cells. Furthermore, even trace amounts of RNases are able to degrade RNA. Note that RNAses are present everywhere (skin, reagents, normal plastic etc).

Therefore, it is essential to avoid inadvertently introducing RNases into the RNA sample during or after the isolation procedure.

Remember that following microbiological aseptical techniques is usually good practice (and a requirement) when working in a molecular biology lab.

There are excellent guides available online for working with RNA:

I took the above paragraph from here: http://www.bioline.com/us/rna-hints-and-tips

Life tech has a few more tips for you: https://www.lifetechnologies.com/uk/en/home/references/ambion-tech-support/nuclease-enzymes/general-articles/working-with-rna.html

Here is a good pdf to have in your labbook as a reference sheet:http://genomics.no/oslo/uploads/PDFs/workingwithrna.pdf

If this scares you , that's good because when working with RNA there are a million things that can go wrong the first few times you try it. You have to be very careful, and adapting a cavalier attitude such as not bothering is going to come back and haunt you at night...

  • $\begingroup$ I tend to disagree. What use will 70% ethanol have on most contaminations? Mostly none, and I even doubt that it will inhibit bacteria (as people only spray it on or do a short wipe). $\endgroup$ – Chris May 23 '15 at 23:07
  • $\begingroup$ @Chris What I meant was for surface cleansing. We tend to spray the ethanol and wait a few minutes and then wipe it out ? I was always told that ethanol denatured proteins ? $\endgroup$ – Rover Eye May 23 '15 at 23:20
  • $\begingroup$ I keep the surface of my bench clean, so I don't need any extra cleaning before I start working. And yes, ethanol can denaturate proteins, but I wouldn't rely on this. Besides that: Your reaction will take place inside plastic tubes which are clean. $\endgroup$ – Chris May 23 '15 at 23:41
  • $\begingroup$ @Chris true that, but I always assume the worst. I was trained in cell biology before mol biology, and yeah... suffice to say I my just have a 70% ethanol spray somewhere in my pocket :) $\endgroup$ – Rover Eye May 24 '15 at 0:05
  • $\begingroup$ I do a lot of cell culture, too. And many people spray everything (even the outside of single-packaged plastic pipettes, which will never get into contact with everything. $\endgroup$ – Chris May 24 '15 at 10:06

First of all, sterility is not necessary. It takes much more effort to reach this than just to wipe down everything with ethanol. What you need is a clean and controlled work environment (but this is something you need anyway to get reproducible results) and good and clean equipment. You will need more precautions for RNA work as RNA is more sensitive and RNAses are ubiquituous, but the general rules are the same. Some remarks from me about it:

  • There is no need to wipe down everything in your environment before working. Will you DNA touch the desk or the outside of you pipette? Simply wear gloves, use fresh plasticware, do not touch the plasticware with your bare finger and you will be fine.

  • There is no general need to autoclave everything what you use (unless you explicitly need it sterile). Do not touch the stuff with your bare fingers. Autoclaves can also be a souce for contamination when they are badly maintained or used to also autoclave trash. Plasticware is generally nuclease free, thanks to the production process (melted plastic is not a good place to live on).

  • If you work with RNA try to set up a separated working space to avoid contaminations by your normal work. If you use glassware, wash it with DEPC treated water and autoclave it to get it RNAse free. Use gloves! Also use a different set of chemicals for this work to avoid cross-contamination. Filter-tips can be a good idea here. Besides being careful, RNA work is no magic but solid handcraft.

  • If you do PCR try to set up a different work place seperated from your normal work. PCR is very sensitive to contaminations sitting in you pipettes from your normal work. I have seen a number of cases of false positive amplifications from normal work.

  • $\begingroup$ Re #1: Many people work with DNA without wearing gloves (including touching the plastic ware), and it rarely causes problems if your tips and the insides of your tubes are clean. Re #4: In many cases (cloning or synthetic biology), it is extremely unlikely that the contaminant would have the right primer annealing sequences to compete with your intended reaction. See for instance Colony PCR, a very "dirty" PCR full of "contaminants". $\endgroup$ – Superbest May 24 '15 at 0:30
  • $\begingroup$ @Superbest True. But I have also seen people working with sequences (which they cloned) and then used the same set of pipettes to search for the same sequence. It took a while for them to find out that they managed to contaminate their pipettes through inproper pipetting. But otherwise I agree. $\endgroup$ – Chris May 24 '15 at 10:05

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