All proteins were expressed in E. coli BL21 (DE3).14 (link),18 (link) Enhanced clamp binding mutants of polymerase and exonuclease were used with amino acid changes for the polymerase in residues QADMF to QLDLF from 920–924 and for the exonuclease QTSMAF to QLSLPL from 182–187.14 (link) Pol IIIα, β and ε subunits were expressed and purified as previously described19 (link) and were flash frozen in liquid nitrogen and stored at −80 °C. θ mutant E41C was created to incorporate a Cys residue for maleimide labeling. The Quikchange lightning kit (Agilent) was used for site-directed mutagenesis. θ was purified on a Histrap HP column and a Superdex 75 gel filtration column. The θ subunit containing a E41C mutation was labeled with Cy5 maleimide (GE Healthcare) following manufacturer instructions. Pol III core complexes consisting of α, β and ε were assembled at equistoi-chiometric ratio and then chromatographically separated by gel filtration (Superdex 75 column). Proteins were flash frozen in liquid nitrogen and stored at −80 °C until needed.
Protocol detail
Reconstitution of E. coli DNA Polymerase III Complex
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.
Amino acid
E coli
Exonuclease
Frozen
Gel filtration
Maleimide
Mutation
Nitrogen
Proteins
Site directed mutagenesis
Subunit
Corresponding Organization : MRC London Institute of Medical Sciences
Other organizations : University of North Texas, MRC Laboratory of Molecular Biology
Top 5 similar protocols
Variable analysis
independent variables
- Enhanced clamp binding mutants of polymerase and exonuclease with amino acid changes: for the polymerase in residues QADMF to QLDLF from 920-924, and for the exonuclease QTSMAF to QLSLPL from 182-187
- Protein expression and purification of Pol IIIα, β, and ε subunits
- All proteins were expressed in
E. coli BL21 (DE3) - Pol III core complexes consisting of α, β and ε were assembled at equistoi-chiometric ratio and then chromatographically separated by gel filtration (Superdex 75 column)
- θ mutant E41C was created to incorporate a Cys residue for maleimide labeling
- The θ subunit containing a E41C mutation was labeled with Cy5 maleimide (GE Healthcare) following manufacturer instructions
- 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!