In a previous article, we discussed the basic characteristics of biofilm samples and factors that influence sample prep and handling. Today we want to share with you some very important tips for isolation of DNA or RNA from biofilm samples. After working with numerous different biofilms and biomats, these recommendations are based on our experience and the experiences of the scientists we worked with while developing the PowerBiofilm kit.
This list may expand over time as we continue to learn more about biofilm and work with new and interesting samples. We are always looking for new samples to try in our labs. If you have a sample that is very difficult to work with, let us know and we can try it out and make some custom recommendations.
Here is our current list of Do’s and Don’ts for working with biofilm:
1) DON’T use too much sample. When working in a mini-prep format with 2 ml bead tubes, the recommended sample size range is 0.05 to 0.2 g. While some researchers have successfully used more (0.25 – 0.3 g) this was optimized within their own laboratories. Using more than the recommended sample volume can and often will result in no yield (see also point 3). This sample range is provided not as a guideline but as a range in which the lysis chemistry is optimized. Using more than the recommended sample size will prevent optimal matrix treatment and cell lysis.
2) DO use the bead tubes provided in the kits. The PowerBiofilm Bead Tubes have been specially formulated to work with the lysis chemistry. It’s not just a simple bead mixture. The tube itself is a tough tube so it can be used on the vortex and a high powered bead beater without risk of breaking. Resist the temptation to transfer the beads to a different tube. This may result in components being left behind and incomplete removal of polysaccharide. We know that some people haven’t used opaque bead tubes before, but transferring the homogenate is easy because the debris packs down after lysis. Try it and if you have any problems or concerns, just call us.
3) DON’T homogenize for too long. Using your laboratory’s standard bead beating settings may not be ideal- it actually may be too much! In our experience, beating biofilm samples longer or harder does not improve yield. The longer and harder you homogenize, the finer the polysaccharides and other organic/inorganic material becomes, causing a thickening of the lysate. Much of this material is not soluble and traps nucleic acid, resulting in its loss. If you are removing less than 400 µl of lysate after bead beating using the Powerbiofilm kit, then you may have bead beat for too long. Beating for 30 seconds at a high setting is a good starting point.
4) DO elute in the proper volume. This rule applies to the silica spin filters used for purification. The optimal elution volume is 100 µl. This enables the maximum amount of nucleic acid to be released from the spin column membrane. The minimum amount is 50 µl. If applied evenly to the membrane then you can still obtain your nucleic acid with high efficiency, however, 100 µl ensures a complete recovery. Eluting in less than 50 µl will seriously impact your yield. Remember you can always concentrate your sample after elution if you need a smaller volume. If you need help or a protocol, contact us.
5) DON’T assume that all biofilms and biomats are the same. Some biofilms are more matter and less microbe so the yields may not be as high as you expect. If you don’t see measureable DNA or RNA on a Nanodrop after elution, and you were careful not to use too much and not to over-homogenize, it may still be present in a very low concentration. You should give the PCR a try (see point 6). When in doubt about your biofilm sample and expected yield, contact our Technical Services (firstname.lastname@example.org), where we can likely provide additional optimization steps. For more information on typical yields from different biofilms and biomats, click here.
6) DO evaluate your nucleic acid on a gel or by PCR. When measuring yield using UV, a number of things can influence readings. Humic substances and co-eluting RNA can inflate A260 values significantly. Additionally, sheared DNA will give higher readings than intact DNA. It’s always a good idea to look at your nucleic acid on a gel to make sure that yields as measured on a spec are really accurate. Because the PowerBiofilm method uses Inhibitor Removal Technology (IRT), it is very pure and so the readings, while low, are most likely accurate compared to methods that do not sufficiently clean the DNA or RNA. But if your yields are too small to see on a gel, then try PCR. The incorporation of IRT in the protocol will enable amplification out of biofilm samples that would fail using other methods.
We hope this list of technical tips for working with biofilms is a help to all of you struggling to get molecular information from these precious samples. We know how much time and effort (and money) goes into the field trips for collecting biofilm and biomat and we want you to be successful. More technical tips will be posted over time. We welcome you to share with us some of your tips and tricks for biofilm work.
If we can help you specifically with your biofilm or biomat sample, let us know at email@example.com
~Suzanne and Heather