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Team:Goettingen/protocol Plasmid Con
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Incubate reaction at 25°C for 1 hour. <br /> | Incubate reaction at 25°C for 1 hour. <br /> | ||
Add 2 μl of 2 μg/μl Proteinase K solution and incubate at 37°C for 10 minutes. <br /> | Add 2 μl of 2 μg/μl Proteinase K solution and incubate at 37°C for 10 minutes. <br /> | ||
| - | Transform competent <i>E. coli</i> and select for the appropriate antibiotic-resistant expression clones. | + | Transform competent <i>E. coli</i> and select for the appropriate antibiotic-resistant expression clones.<br /><br /> |
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Revision as of 13:43, 13 October 2014
Overview
PCR Methods
Plasmid Construction
- Restriction of DNA
- Ligation of DNA fragments
- BP recombination reaction
- LR recombination reaction
- SEAMLESS Cloning
- Peptide Library construction
Plasmid Transformation
- E.coli competent cells
- Plasmid isolation (E.coli)
- E.coil transformation
- Plasmid isolation (Yeast)
- Yeast transformation
Colony Scanning
Protein Assessment
In vivo tests
BP recombination reaction
Perform a BP recombination reaction between an attB-flanked DNA fragment and an attP-containing donor vector to generate an entry clone.
Add the following components to a 1.5 ml microcentrifuge tube at room temperature and mix:
attB-PCR product 0.6 μl
pDONR™ vector (supercoiled, 150 ng/μl) 0.2 μl
Vortex BP Clonase™ enzyme mix briefly.
Add 0.2 μl to the components above and mix well by vortexing briefly twice.
Immediately back into -80°C!
Incubate reaction at 25°C for 1 hour.
Add 2 μl of 2 μg/μl Proteinase K solution and incubate at 37°C for 10 minutes.
Transform competent E. coli and select for the appropriate antibiotic-resistant entry clones.
LR recombination reaction
Perform an LR recombination reaction between an attL-containing entry clone and an attR-containing destination vector to generate an expression clone.
Add the following components to a 1.5 ml microcentrifuge tube at room temperature and mix:
Entry clone (supercoiled, 100-300 ng) 0.6 μl
Destination vector (supercoiled, 150 ng/μl) 0.2 μl
Vortex LR Clonase™ enzyme mix briefly. Add 0.2 μl to the components above and mix well by vortexing briefly twice.
Immediately back into -80°C!
Incubate reaction at 25°C for 1 hour.
Add 2 μl of 2 μg/μl Proteinase K solution and incubate at 37°C for 10 minutes.
Transform competent E. coli and select for the appropriate antibiotic-resistant expression clones.
