In vitro NHEJ assays were performed essentially as described previously (19 (link)). Briefly, reactions (10 μl) were performed in 50 mM HEPES pH 8.0, 100 mM KOAc, 0.5 mM Mg(OAc)2, 1 mM ATP, 1 mM DTT and 0.1 mg/ml bovine serum albumin (BSA) and the indicated concentration of Ku-DBi. pBluescript DNA was linearized with HindIII and 5′-32P-labeled. Reactions contained 10 ng of DNA and 25 μg of whole cell extract. Reactions were incubated at 37°C for 2 h, terminated by the addition of proteinase K/SDS/EDTA and incubated for an additional 30 min at 37°C. Products were separated by electrophoresis on 0.6% agarose gels. Products were detected in dried gels by PhosphorImager (BioRad PMI) analysis.
Protocol detail
In Vitro NHEJ Assay Protocol
In vitro NHEJ assays were performed essentially as described previously (19 (link)). Briefly, reactions (10 μl) were performed in 50 mM HEPES pH 8.0, 100 mM KOAc, 0.5 mM Mg(OAc)2, 1 mM ATP, 1 mM DTT and 0.1 mg/ml bovine serum albumin (BSA) and the indicated concentration of Ku-DBi. pBluescript DNA was linearized with HindIII and 5′-32P-labeled. Reactions contained 10 ng of DNA and 25 μg of whole cell extract. Reactions were incubated at 37°C for 2 h, terminated by the addition of proteinase K/SDS/EDTA and incubated for an additional 30 min at 37°C. Products were separated by electrophoresis on 0.6% agarose gels. Products were detected in dried gels by PhosphorImager (BioRad PMI) analysis.
Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link:
Access Free Full Text.
Corresponding Organization : NĒRx BioSciences (United States)
Other organizations : University of Illinois Urbana-Champaign, University of Illinois at Chicago, Illinois College
Top 5 similar protocols
Variable analysis
independent variables
- Concentration of Ku-DBi
- NHEJ (non-homologous end joining) activity
- 50 mM HEPES pH 8.0
- 100 mM KOAc
- 0.5 mM Mg(OAc)2
- 1 mM ATP
- 1 mM DTT
- 0.1 mg/ml bovine serum albumin (BSA)
- Linearized pBluescript DNA with HindIII
- 25 μg of whole cell extract
- Positive control: Not explicitly mentioned.
- Negative control: Not explicitly mentioned.
Annotations
Based on most similar protocols
Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.
Sign up for free or login to display all annotations
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!