The concentrated samples were divided into two aliquots and one of the aliquots was treated with 12.5 μL of PMA (2 mM; Biotium, Inc., Hayward, CA) to a final concentration of 25 μM [80 (link)], followed by thorough mixing and incubation in the dark for 5 min at room temperature. Samples were then exposed to light with the PhAST blue-photoactivation system for tubes (GenIUL, S.L., Terrassa, Spain) for 15 min [81 (link)]. Information deduced from PMA-treated samples was documented for viable microbial population and data derived from the PMA-untreated aliquot was reported as total (dead and live) microbial population. Both, the PMA-treated and non-treated samples were further split in half, with one half subjected to bead beating with the Fastprep-24 bead-beating instrument (MP Biomedicals, Santa Ana, CA). The samples were run at 5 m/s for 60 s. After bead beating, the samples were combined with their respective analog, which were not subjected to bead beating, and the DNA from the combined sample was extracted by the Maxwell-16 MDx automated system according to the manufacturer’s instructions (Promega, Madison, WI). The purified DNA was eluted into a final volume of 50 μL.
Fastprep 24 bead beating instrument
Manufactured by MP Biomedicals
Sourced in United States
The Fastprep-24 is a bead-beating instrument used for the rapid and efficient homogenization of samples. It is designed to effectively disrupt a wide range of sample types, including tissues, cells, and microorganisms, to facilitate the extraction of nucleic acids, proteins, and other biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Phorbol myristate acetate (pma), by Biotium (2 mentions)
Maxwell 16 mdx automated system, by Promega (1 mentions)
Phorbol myristate acetate (pma), by MP Biomedicals (1 mentions)
Maxwell 16 tissue lev total rna purification kit, by Promega (1 mentions)
Co2 multitron incubator, by Infors (1 mentions)
Lysing matrix a, by MP Biomedicals (1 mentions)
4 protocols using fastprep 24 bead beating instrument
1
Viable Microbial Population Analysis
2
Preparing DNA from Microbial Samples
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Both antibiotic-treated and -untreated samples were divided into equal aliquots. One half of each set was treated with PMA dye (final concentration 25 μM) (Biotum, Inc., Hayward, CA) and the other half was left untreated following exactly the steps described elsewhere (Blachowicz et al., 2017 (link)). In brief, PMA-treated and untreated samples were kept in darkness for 5 min, followed by 15 min exposure to light in PHaST Blue-Photo activation system for tubes (GenIUL, S.L, Terrassa, Spain). Following dye intercalation, each sample was split in half. One aliquot was subjected to bead-beating for 60 s at 5 m/s on the Fastprep-24 bead-beating instrument (MP Biomedicals, Santa Ana, CA). Subsequently, both bead-beaten and not bead-beaten aliquots were combined and used for DNA extraction using Maxwell 16 Tissue LEV Total RNA purification kit following manufacturer’s instructions (Promega, Madison, WI). As previously reported, the DNA purification was based on magnetic beads capturing and washing off non-DNA materials (Venkateswaran et al., 2012 ). Extracted and purified DNA was divided into aliquots and stored at −80°C until further analysis.
3
Transient Transfection of CHO Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CHO cells were transiently transfected, as described recently [21 (link)]. Briefly, 1.5 μg pcDNA3.1-eIF2α-S52A or pCAG-T7pol expression plasmid/106 cells and 2 μg PEI reagent/106 cells were incubated for 4 h at a cell density of 4 × 106 cells/ml at 37 °C, 5% CO2 and 80 rpm in the CO2 Multitron incubator (Infors). Afterwards, 150 ml fresh culture medium was added to achieve a 200 ml culture at a cell density of 106 cells/ml for two days at 37 °C, 5% CO2 and 100 rpm. CHO cells were harvested and washed, as described previously [14 (link)]. Cell lysate was prepared by using the lysing matrix A (MP Biomedicals). The wet cell pellet was transferred to a lysing matrix A tube and cells were disrupted for 5 s and 4 m/s in the presence of dry ice in the cooling chamber of the FastPrep-24 Bead-Beating instrument (MP Biomedicals) and cell lysates were isolated as described previously [14 (link)].
4
Microbial DNA Extraction and Viability Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All filtered samples were then divided into three separate aliquots: one of the aliquots (500 μl) was subjected to PMA pretreatment (viability assessment), the second (500 μl) was an untreated environmental sample (viable + nonviable cells; total DNA), and the third (500 μl) was used for ATP analysis (see below). One 500 μl aliquot of filter-concentrated sample suspension was treated with PMA (Biotium, Inc., Hayward, CA, USA) to a final concentration of 50 μM [27 (link),31 (link)], followed by thorough mixing and incubation in the dark for 5 min at room temperature. The sample was exposed to PhAST blue-Photo activation system for tubes (GenIUL, S.L., Terrassa, Spain) for 15 min (in parallel with the non-PMA treated sample). Samples were then split in half and one half was subjected to bead beating with the Fastprep-24 bead-beating instrument (MP Biomedicals, Santa Ana, CA, USA) with parameters set at 5 m/s for 60 s. The second half of the unprocessed sample was then combined with the mechanically disrupted counterpart to allow microbial yields from hardy cells and spores with a limited loss of the overall microbial diversity. DNA was extracted via the Maxwell 16 automated system (Promega, Madison, WI, USA), in accordance with manufacturer’s instructions, and resulting DNA suspensions (100 μl each) were stored at −20°C.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!