DNA plugs for Pulse-Field Gel Electrophoresis analysis were prepared using a previously described method (Yanagi et al., 2022) as follows. In this study, cells were inoculated directly from stock into liquid medium and incubated until saturation, except when a single colony was isolated. Cells from the saturated culture medium (5 × 107 cells per plug) were collected and washed twice with 50 mM EDTA (pH 7.5). For each agarose plug, washed cells were resuspended in 33 μL of 50 mM EDTA (pH 7.5) and then mixed with 66 μL of solution I containing 8.3 mg/mL low-melting-point agarose SeaPlaque GTG (Lonza), 170 mM sorbitol, 17 mM sodium citrate, 10 mM EDTA (pH 7.5), 0.85% v/v b-mercaptoethanol, and 0.17 mg/mL Zymolyase 100 T (Nacalai). The solution was vortexed, poured into plug molds (Bio-Rad), and solidified at 4°C. Plugs were treated with a solution II containing 450 mM EDTA (pH 7.5), 10 mM Tris-HCl (pH 7.5), 7.5% v/v b-mercaptoethanol, and 10 mg/mL RNaseA (Macherey-Nagel) for 1 to 1.5 h at 37°C. Then, solution II was discarded and replaced with solution III containing 250 mM EDTA (pH 7.5), 10 mM Tris-HCl (pH 7.5), 10 g/L SDS and 1 mg/mL Proteinase K (Merck Millipore). The plugs were then incubated overnight at 50°C, washed four times with 50 mM EDTA (pH 7.5), and stored at 4°C.
Seaplaque gtg
Manufactured by Lonza
SeaPlaque GTG is a high-quality laboratory equipment product manufactured by Lonza. It is designed for use in a variety of scientific applications, providing a reliable and consistent performance. The core function of this product is to facilitate the growth and enumeration of microorganisms in a controlled environment.
Automatically generated - may contain errors
2 protocols using seaplaque gtg
1
Preparation of DNA Plugs for PFGE
2
Preparation of Genomic DNA Plugs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For PFGE, ERC, DSB, and 2D gel analyses, genomic DNA was prepared in low melting temperature agarose plugs as described previously (Sasaki and Kobayashi, 2017; 2021) (link). Briefly, collected cells were resuspended in 50 mM EDTA pH 7.5 (33 μL cell per 5 × 10 7 cells and incubated at 42°C. For each plug, 33 μL cell suspension was mixed with 66 μL solution 1 (0.83% low-melting-point agarose SeaPlaque GTG (Lonza), 170 mM sorbitol, 17 mM sodium citrate, 10 mM EDTA pH 7.5, 0.85% β-mercaptoethanol, and 0.17 mg/mL Zymolyase 100 T (Nacalai)), poured into a plug mold (Bio-RAD) and placed at 4°C for the agarose to be solidified. Plugs were transferred to a 2 mL-tube containing solution 2 (450 mM EDTA pH 7.5, 10 mM Tris-HCl pH 7.5, 7.5% β-mercaptoethanol and 10 μg/mL RNaseA (Macherey-Nagel)), and incubated for 1 h at 37°C. Plugs were then incubated overnight at 50°C in solution 3 (250 mM EDTA pH 7.5, 10 mM Tris-HCl pH 7.5, 1% sodium dodecyl sulfate (SDS) and 1 mg/mL Proteinase K (Nacalai)).
Plugs were washed four times with 50 mM EDTA pH 7.5 and stored at 4°C in 50 mM EDTA pH 7.5.
Plugs were washed four times with 50 mM EDTA pH 7.5 and stored at 4°C in 50 mM EDTA pH 7.5.
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!