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Molecular Cloning
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Molecular Cloning
Contents |
Plasmids
1 pUCPhbCAB
2 pACYC(lacI+) DuetSRRz
3 pACYC(lacI+) DuetEGFP
4 pACYC(lacI-) DuetSRRz
5 pACYC(lacI-) DuetEGFP
6 pACYC(LacI-)DuetSRRzPpPhaPLacILVACret7terPrPhaRLoxPLacILoxP
7 pACYC(LacI-)DuetEGFPPpPhaPLacILVACret7terPrPhaRLoxPLacILoxP
Template Resources
PhbCAB: PBHR68 Plasmid
Lac I: PET28a
CRE:
PpPhaP: Ralstonia eutropha H16 genome
PrPhaR: Ralstonia eutropha H16 genome
SRRz:
EGFP:
Primers
(1) SRRz Primers
SRRZ-NcoI-for
5- ATCCATGGATGAAGATGCCAGAAAAACATGACCTGTTG-3
SRRZ-rev-in
5 - ACCCCGCCGAAGCGGGGTTTTTTTTTCTACTATCTGCACTGCTCATTAATA-3
SRRZ-BamHI-rev-out
5 -TATGGATCCAAAAAAAAACCCCGCCGAAGCGGGGTT-3
(2) Pp Operon primers
PP-PhaP-NotI-for
5- TATGCGGCCGCTGTTTGTGCATTGCACAAAATCCA-3
PP-PhaP-HindIII-rev
5- CACCATGTCGACTTTCTCCTCTTTAAGCTTTCAGGCAGCCGTCGTCTTCTTTG-3
LacILVA-HindIII-for
5-TGCCTGAAAGCTTAAAGAGGAGAAAGTCGACATGGTGAATGTGAAACCAGTAAC-3
LacILVA-rev-in
5- AGCTACTAAAGCGTAGTTTTCGTCGTTTGCAGCCTGCCCGCTTTCCAGTCGGGAAACCT-3
LacILVA-rev-PstI-out
5- ATTGGACATGCGCGCTTTCTCCTCTTTCTGCAGTTATTAAGCTACTAAAGCGTAGTTTT-3
IGEM-ZouYLP-CreT7ter—PstI-for
5- TGCAGAAAGAGGAGAAAGCGCGCATGTCCAATTTACTGACCGTACACCAAAATT-3
IGEM-ZouYLP-CreT7ter-rev-in
5-AGCGGGGTTTTTTTTTCTACTAATCGCCATCTTCCAGCAG-3
IGEM-ZouYLP-CreT7ter-EcoRI-rev-out
5-ATGAATTCGAGCTCGAAAAAAAAACCCCGCCGAAGCGGGGTTTTTTTTTCTACTAAT-3
(3) Pr operon primers
PR-PhaR-NotI-for
5-ATGCGGCCGCAGTGCCTTGTTGGGCATAGAATCAGGGCAGCGGCGCAGC-3
PR-PhaR-NdeI-rev
5-ACGAAGTTATCATATGTTATTACTTCTTGTCCGGCTGGTTGAACGGGAACGTCCCGAAC-3
LRLacILP-for-in
5-TGCTATACGAAGTTATAAAGAGGAGAAACTCGAGATGGTGAATGTGAAACCAGTAACGT-3
LRLacILP-NdeI-for-out
5-ACAAGAAGTAATAACATATGATAACTTCGTATAATGTATGCTATACGAAGTTATAAAGA-3
LRLacILP-rev-in
5-TACGAAGTTATCTACTGCCCGCTTTCCAGTCGGGAAACCT-3
LRLacILP-KpnI-rev-out
5-ATGGTACCATAACTTCGTATAGCATACATTATACGAAGTTATCTACTGCCCGCTT-3
(4) EGFP Primers
EGFP-for-KoZg-for
5-ccatgggcagcaagggcgaggagctgttc-3
EGFP-rev-in
5-AAAAAAAAACCCCGCCGAAGCGGGGTTTTTTTTTCTATCACTTGTACAGCTCGTC-3
EGFP-rev-out
5-TTTTC GAGCTC GAATTC GGATCCAAAAAAAAACCCCGCCGAA-3
Fragment Preparation
SRRz: PCR with primer SRRZ-NcoI-for and SRRZ-rev-in;
Purify the product and run PCR with primer SRRZ-NcoI-for and primer SRRZ-BamHI-rev-out;
EGFP: PCR with primer EGFP-for-KoZg-for and EGFP-rev-in;
Purify the product and run PCR with primer EGFP-for-KoZg-for and primer EGFP-rev-out;
PpPhaP: PCR with primer PP-PhaP-NotI-for and PP-PhaP-HindIII-rev;
LacI(LVA): PCR with primer LacILVA-HindIII-for and LacILVA-rev-in;
Purify the product and run PCR with primer LacILVA-HindIII-for and primer LacILVA-rev-PstI-out;
CRE(T7ter): PCR with primer CreT7ter—PstI-for and CreT7ter-rev-in
Purify the product and run PCR with primer CreT7ter—PstI-for and CreT7ter-EcoRI-rev-out;
PrPhaR: PCR with primer PR-PhaR-NotI-for and PR-PhaR-NdeI-rev;
LoxPLacILoxP: PCR with primer LRLacILP-for-in and LRLacILP-rev-in
Purify the product and run PCR with primer LRLacILP-NdeI-for-out and primer LRLacILP-KpnI-rev-out.
Fusion PCR
Cut and Insert
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