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Team:Goettingen/protocol Plasmid Con
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Transform competent <i>E. coli</i> and select for the appropriate antibiotic-resistant expression clones.<br /><br /> | Transform competent <i>E. coli</i> and select for the appropriate antibiotic-resistant expression clones.<br /><br /> | ||
</p> | </p> | ||
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| + | <h3 id="LB">Peptide Library Construction</h3> | ||
| + | <p>Starting with GFP scaffolds (Denoted pRS 316-GFPM), random loop libraries were created. Randomized amino acids were inserted at Asp-102/Asp-103 and Glu-172/Asp-173 by using the NNK method in order to create the random loop libraries. NNK sequences in primers number 13 and 16 were used to create regions I and ӀӀӀ. After having randomized DNA sequences, these two regions were assembled with region ӀӀ which remained intact. In each of the amplification and extension steps, <a href="https://2014.igem.org/Team:Goettingen/protocol_PCR#Pfus_PCR"><i>Pfus</i> PCR protocol</a> was used. For the assembly of the three regions, an <a href="https://2014.igem.org/Team:Goettingen/protocol_PCR#Asse_PCR">assembly PCR</a> was used. <br /><br /> | ||
| + | </p> | ||
| + | <center><img src="https://static.igem.org/mediawiki/2014/d/d3/Goettingen_peptide_library.png" width="350px"/></center><br /> | ||
| + | <p style="text-align:center"><b>Figure. Peptide Library Construction</b></p> <br /><br /> | ||
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Revision as of 20:52, 15 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.
Peptide Library Construction
Starting with GFP scaffolds (Denoted pRS 316-GFPM), random loop libraries were created. Randomized amino acids were inserted at Asp-102/Asp-103 and Glu-172/Asp-173 by using the NNK method in order to create the random loop libraries. NNK sequences in primers number 13 and 16 were used to create regions I and ӀӀӀ. After having randomized DNA sequences, these two regions were assembled with region ӀӀ which remained intact. In each of the amplification and extension steps, Pfus PCR protocol was used. For the assembly of the three regions, an assembly PCR was used.
Figure. Peptide Library Construction
