Introduction of biological film DNA and RNA extraction points

Earlier articles we discussed the basic properties of biofilm samples and the factors that influence sample preparation and processing. Today we share with you a few key points in extracting DNA or RNA from biofilm samples. Below is a summary of our extensive work on various types of biofilms and biomats, as well as the experience of scientists working with us to develop the PowerBiofilm Kit.

The list below may increase as the biofilm continues to be studied in depth or encounters new types of interesting samples. If your sample is very difficult to handle, contact us or give you a good suggestion.

1) Add less. When using a small amount of 2ml Bead Tubes for extraction, the loading amount was controlled at 0.05-0.2 g. Some researchers have long-term exploration and optimization through their own laboratories, and the amount of sample loading has reached 0.25-0.3g. Use of more than the recommended amount of loading often results in the inability to raise nucleic acids (see point 3). It is recommended that the amount of sample added is not the scope of guidance but the optimal treatment range for the lysis solution chemistry. Excessive loading can affect the removal of biofilm growth matrices and cell lysis.

2) Use a kit to provide a beaded bead. PowerBiofilm's abrasive bead casings are specifically designed to work with cleavage solution chemicals. It is not a simple mixing of beads. The outer container Tube itself is very hard and can be applied to ordinary vortex oscillations and powerful high-speed bead grinding machines without rupture in the middle. Please do not transfer the beads to other Tube tubes. Some people have never used an opaque grinding bead casing, but after centrifugation, the debris will precipitate, and it will not be difficult to transfer the supernatant. Try it, if you have any difficulties, please feel free to contact us.

3) Do not time out grinding. It is not always best to use your laboratory's conventional grinding homogenization equipment and standard settings, and it is possible to over-grind. According to our experience, lengthening the sample grinding time or greater grinding strength does not increase the yield. As the milling time increases and the strength increases, polysaccharides and other organic/inorganic components tend to form viscous lysates. Most of these substances are insoluble and will adsorb nucleic acids, resulting in loss of nucleic acids. If the transferred lysate after grinding is less than 400 μl, then the grinding is too much. High-speed grinding for 30s is more suitable.

4) Use an appropriate amount of eluent. This applies to the elution using a silica filter column. The best eluent volume is 100 μl. This amount allows the nucleic acid to elute sufficiently. Evenly added to the filter, the minimum amount of 50 μl also ensures the efficiency of nucleic acid elution, but 100 μl can obtain the most elution. Use of less than 50 μl can seriously affect nucleic acid yield. Keep in mind that diluted nucleic acids can be reduced in size by concentration.

5) Do not assume that all biofilms and biomats are similar. Some biofilms contain very few microbes, and the yield is far less than expected. If the spectrophotometer does not detect DNA or RNA after elution, the amount of sample does not exceed the recommended value, and there is no grinding too much, perhaps only the nucleic acid concentration is very low. You still have the possibility to detect it by PCR (see point 6). If you don't know much about your biofilm sample and the expected yield is very different, please contact us or give more optimization suggestions. For general yields of some biofilms and biopads, click here (biofilm_apnote).

6) Gel electrophoresis and PCR jointly evaluate nucleic acids. Using UV light to detect yields, many factors can affect the accuracy of the readings. Humic acid and contaminating RNA can significantly push up A260. DNA fragments are higher than the complete DNA fragment readings. Gel electrophoresis can better test the results of the spectrophotometer. Because PowerBiofilm uses IRT technology (inhibitor removal technology), the nucleic acids obtained are very pure, so if the readings are lower, it is more accurate than the results obtained without efficient purification of DNA and RNA. If the band is not visible by gel electrophoresis, try PCR amplification. The PowerBiofilm Kit using IRT technology guarantees the success rate of amplification, which is significantly different from other extraction methods.

summary:

I hope that the above suggestions will help you to work hard to extract the biological information of this precious sample. Spending a lot of time and money on long journeys to sample hard and tired, we understand! I hope that your experiment will be successful. More technical tips will be released later. You are also welcome to discuss the experience and skills of biofilms with our risk.