Neutral–neutral 2D gel electrophoresis was performed according to a previous description [21 (link)]. Typically, 5–10 μg of BND cellulose-enriched RIs was loaded on a 0.4% agarose gel, and the one-dimensional gel was run in 1×TAE buffer at 1.5 V/cm for 18 h at RT. The gel was visualized by ethidium bromide staining, and a gel slice from each lane was cut with the correct size range of RIs and rotated 90 degrees for two-dimensional gel electrophoresis. Gel slices were inserted into 1% agarose gel, and the two-dimensional gel electrophoresis was performed in 1×TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.0) containing 0.5 μg/mL of ethidium bromide at 3 V/cm for 18 h at 4 °C. Southern blot analysis was carried out to detect the patterns of RIs as previously described [21 (link)].
Isolation and Analysis of Replication Intermediates
Neutral–neutral 2D gel electrophoresis was performed according to a previous description [21 (link)]. Typically, 5–10 μg of BND cellulose-enriched RIs was loaded on a 0.4% agarose gel, and the one-dimensional gel was run in 1×TAE buffer at 1.5 V/cm for 18 h at RT. The gel was visualized by ethidium bromide staining, and a gel slice from each lane was cut with the correct size range of RIs and rotated 90 degrees for two-dimensional gel electrophoresis. Gel slices were inserted into 1% agarose gel, and the two-dimensional gel electrophoresis was performed in 1×TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM EDTA, pH 8.0) containing 0.5 μg/mL of ethidium bromide at 3 V/cm for 18 h at 4 °C. Southern blot analysis was carried out to detect the patterns of RIs as previously described [21 (link)].
Corresponding Organization : Texas A&M University
Other organizations : Shanghai Jiao Tong University, Layton BioScience (United States)
Variable analysis
- Restriction enzyme digestion of genomic DNA at 37 °C for 4 h
- Patterns of replication intermediates (RIs) detected by Southern blot analysis
- Total genomic DNA preparation as previously described [21]
- Benzoylated naphthoylated DEAE (BND)-cellulose purification of RIs
- Neutral–neutral 2D gel electrophoresis of RIs
- Ethidium bromide staining and visualization of gels
- Positive control: Not specified
- Negative control: Not specified
Annotations
Based on most similar protocols
As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.
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!