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Team:Goettingen/protocol peptide
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<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 assembly PCR was used. <br /><br /> | <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 assembly PCR was used. <br /><br /> | ||
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| - | <center><img src="https://static.igem.org/mediawiki/2014/d/d3/Goettingen_peptide_library.png" width=" | + | <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> | <p style="text-align:center"><b>Figure. Peptide Library Construction</b></p> | ||
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Revision as of 14:38, 4 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
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
