8: Ligation of a restriction enzyme digested plasmid DNA
- Page ID
- 169772
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\(\newcommand{\avec}{\mathbf a}\) \(\newcommand{\bvec}{\mathbf b}\) \(\newcommand{\cvec}{\mathbf c}\) \(\newcommand{\dvec}{\mathbf d}\) \(\newcommand{\dtil}{\widetilde{\mathbf d}}\) \(\newcommand{\evec}{\mathbf e}\) \(\newcommand{\fvec}{\mathbf f}\) \(\newcommand{\nvec}{\mathbf n}\) \(\newcommand{\pvec}{\mathbf p}\) \(\newcommand{\qvec}{\mathbf q}\) \(\newcommand{\svec}{\mathbf s}\) \(\newcommand{\tvec}{\mathbf t}\) \(\newcommand{\uvec}{\mathbf u}\) \(\newcommand{\vvec}{\mathbf v}\) \(\newcommand{\wvec}{\mathbf w}\) \(\newcommand{\xvec}{\mathbf x}\) \(\newcommand{\yvec}{\mathbf y}\) \(\newcommand{\zvec}{\mathbf z}\) \(\newcommand{\rvec}{\mathbf r}\) \(\newcommand{\mvec}{\mathbf m}\) \(\newcommand{\zerovec}{\mathbf 0}\) \(\newcommand{\onevec}{\mathbf 1}\) \(\newcommand{\real}{\mathbb R}\) \(\newcommand{\twovec}[2]{\left[\begin{array}{r}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\ctwovec}[2]{\left[\begin{array}{c}#1 \\ #2 \end{array}\right]}\) \(\newcommand{\threevec}[3]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\cthreevec}[3]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \end{array}\right]}\) \(\newcommand{\fourvec}[4]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\cfourvec}[4]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \end{array}\right]}\) \(\newcommand{\fivevec}[5]{\left[\begin{array}{r}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\cfivevec}[5]{\left[\begin{array}{c}#1 \\ #2 \\ #3 \\ #4 \\ #5 \\ \end{array}\right]}\) \(\newcommand{\mattwo}[4]{\left[\begin{array}{rr}#1 \amp #2 \\ #3 \amp #4 \\ \end{array}\right]}\) \(\newcommand{\laspan}[1]{\text{Span}\{#1\}}\) \(\newcommand{\bcal}{\cal B}\) \(\newcommand{\ccal}{\cal C}\) \(\newcommand{\scal}{\cal S}\) \(\newcommand{\wcal}{\cal W}\) \(\newcommand{\ecal}{\cal E}\) \(\newcommand{\coords}[2]{\left\{#1\right\}_{#2}}\) \(\newcommand{\gray}[1]{\color{gray}{#1}}\) \(\newcommand{\lgray}[1]{\color{lightgray}{#1}}\) \(\newcommand{\rank}{\operatorname{rank}}\) \(\newcommand{\row}{\text{Row}}\) \(\newcommand{\col}{\text{Col}}\) \(\renewcommand{\row}{\text{Row}}\) \(\newcommand{\nul}{\text{Nul}}\) \(\newcommand{\var}{\text{Var}}\) \(\newcommand{\corr}{\text{corr}}\) \(\newcommand{\len}[1]{\left|#1\right|}\) \(\newcommand{\bbar}{\overline{\bvec}}\) \(\newcommand{\bhat}{\widehat{\bvec}}\) \(\newcommand{\bperp}{\bvec^\perp}\) \(\newcommand{\xhat}{\widehat{\xvec}}\) \(\newcommand{\vhat}{\widehat{\vvec}}\) \(\newcommand{\uhat}{\widehat{\uvec}}\) \(\newcommand{\what}{\widehat{\wvec}}\) \(\newcommand{\Sighat}{\widehat{\Sigma}}\) \(\newcommand{\lt}{<}\) \(\newcommand{\gt}{>}\) \(\newcommand{\amp}{&}\) \(\definecolor{fillinmathshade}{gray}{0.9}\)Introduction:
DNA ligase of E. coli is a polypeptide of molecular weight 75,000. The comparable T4-induced enzyme is somewhat smaller (63,000 to 68,000). Both enzymes catalyze the synthesis of phosphodiester bonds between adjacent 5'-phosphoryl and 3'-hydroxyl groups in nicked duplex DNA, coupled to the cleavage of the pyrophosphate bond of DPN (E. coli) or ATP (T4). Phosphodiester bond synthesis catalyzed by both enzymes occurs in a series of these discrete steps and involves the participation of two covalent intermediates. A steady state kinetic analysis of the reaction-catalyzed E. coli ligase supports this mechanism, and further demonstrates that enzyme-adenylate and DNA-adenylate are kinetically significant intermediates on the direct path of phosphodiester bond synthesis.
In this experiment, the plasmid DNA pGFPuv (map as shown below) was first cut by NcoI, which disrupt the GFPuv gene. As contrast to the ccc form of plasmid DNA, the linearized plasmid has zero or low transformation efficiency. However, when the linerized plasmid is resealed by T4 DNA ligase, its transformation efficiency will be increased dramatically. The condensed plasmid will have higher transformation efficiency.
Table. Ligation reaction set up
|
1 |
2 |
|
|
Ligation (ul) |
Negative control (no ligase added) (ul) |
|
|
Insert DNA (2-10 ng or in a ratio of 10x or more to vector), for self ligation, add ddH2O |
1 |
1 |
|
Cut vector (25 ng/ul) |
0.5 |
0.5 |
|
2x rapid ligation buffer |
2.5 |
2.5 |
|
T4 DNA ligase (20 U/ul) |
1 |
0 |
|
H2O |
0 |
1 |
|
Total volume (ul) |
5 |
5 |
Mix well and spin briefly
Incubate at room temperature for 5 min to 1 hr.
Use 2.5ul for transformation,
Note:
1. Insert DNA can be a purified gene fragment from gel, or a DNA fragment in low melting gel slice (melt the gel slice at 60C for 2 min before use).
2. For PCR products with a 3’-A , use the TA cloning vector pEZT-T (linearized); for PCR products with blunt ends, use a vector with blunt ends such as pEZT-blunt (CIP treated); for gene fragments with sticky ends, use vector with compatible ends.
3. Transformation efficiency is the number of transformed cells (transformants) generated by 1 µg of supercoiled plasmid DNA in a transformation reaction. A known quantity of plasmid DNA (1 ng) is used as the control.
Transformation efficiency (transformants/µg) is calculated as follows:
# of colonies on plate/ng of DNA plated X 1000 ng/µg
4. 2xrapid ligation buffer: 60 mM Tris-HCl, pH 7.8, 20 mM MgCl2, 20 mM DTT, 2 mM ATP, 10% PEG 8000.
Note: The substrate of ligase is the nicked ds DNA. Two intermediates are present: ligase-adenylate and DNA-adenylate.
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