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Molecular Cloning
From 2008.igem.org
Ylzouyilong (Talk | contribs) (→Primers) |
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| Line 47: | Line 47: | ||
SRRZ-rev-in | SRRZ-rev-in | ||
| - | 5 - ACCCCGCCGAAGCGGGGTTTTTTTTTCTACTATCTGCACTGCTCATTAATA-3 | + | <span style="color: Orange">5 - ACCCCGCCGAAGCGGGGTTTTTTTTTCTACTATCTGCACTGCTCATTAATA-3</span> |
SRRZ-BamHI-rev-out | SRRZ-BamHI-rev-out | ||
| - | 5 -TATGGATCCAAAAAAAAACCCCGCCGAAGCGGGGTT-3 | + | <span style="color: red">5 -TATGGATCCAAAAAAAAACCCCGCCGAAGCGGGGTT-3</span> |
'''(2) Pp Operon primers''' | '''(2) Pp Operon primers''' | ||
| Line 57: | Line 57: | ||
PP-PhaP-NotI-for | PP-PhaP-NotI-for | ||
| - | 5- TATGCGGCCGCTGTTTGTGCATTGCACAAAATCCA-3 | + | <span style="color: Pink">5- TATGCGGCCGCTGTTTGTGCATTGCACAAAATCCA-3</span> |
PP-PhaP-HindIII-rev | PP-PhaP-HindIII-rev | ||
| - | 5- CACCATGTCGACTTTCTCCTCTTTAAGCTTTCAGGCAGCCGTCGTCTTCTTTG-3 | + | <span style="color: Purple">5- CACCATGTCGACTTTCTCCTCTTTAAGCTTTCAGGCAGCCGTCGTCTTCTTTG-3</span> |
LacILVA-HindIII-for | LacILVA-HindIII-for | ||
| - | 5-TGCCTGAAAGCTTAAAGAGGAGAAAGTCGACATGGTGAATGTGAAACCAGTAAC-3 | + | <span style="color: Yellow">5-TGCCTGAAAGCTTAAAGAGGAGAAAGTCGACATGGTGAATGTGAAACCAGTAAC-3</span> |
LacILVA-rev-in | LacILVA-rev-in | ||
| - | 5- AGCTACTAAAGCGTAGTTTTCGTCGTTTGCAGCCTGCCCGCTTTCCAGTCGGGAAACCT-3 | + | <span style="color: Green">5- AGCTACTAAAGCGTAGTTTTCGTCGTTTGCAGCCTGCCCGCTTTCCAGTCGGGAAACCT-3</span> |
LacILVA-rev-PstI-out | LacILVA-rev-PstI-out | ||
| - | 5- ATTGGACATGCGCGCTTTCTCCTCTTTCTGCAGTTATTAAGCTACTAAAGCGTAGTTTT-3 | + | <span style="color: Brown">5- ATTGGACATGCGCGCTTTCTCCTCTTTCTGCAGTTATTAAGCTACTAAAGCGTAGTTTT-3</span> |
IGEM-ZouYLP-CreT7ter—PstI-for | IGEM-ZouYLP-CreT7ter—PstI-for | ||
| - | 5- TGCAGAAAGAGGAGAAAGCGCGCATGTCCAATTTACTGACCGTACACCAAAATT-3 | + | <span style="color: Red">5- TGCAGAAAGAGGAGAAAGCGCGCATGTCCAATTTACTGACCGTACACCAAAATT-3</span> |
IGEM-ZouYLP-CreT7ter-rev-in | IGEM-ZouYLP-CreT7ter-rev-in | ||
| - | 5-AGCGGGGTTTTTTTTTCTACTAATCGCCATCTTCCAGCAG-3 | + | <span style="color: Pink">5-AGCGGGGTTTTTTTTTCTACTAATCGCCATCTTCCAGCAG-3</span> |
IGEM-ZouYLP-CreT7ter-EcoRI-rev-out | IGEM-ZouYLP-CreT7ter-EcoRI-rev-out | ||
| - | 5-ATGAATTCGAGCTCGAAAAAAAAACCCCGCCGAAGCGGGGTTTTTTTTTCTACTAAT-3 | + | <span style="color: Yellow">5-ATGAATTCGAGCTCGAAAAAAAAACCCCGCCGAAGCGGGGTTTTTTTTTCTACTAAT-3</span> |
'''(3) Pr operon primers''' | '''(3) Pr operon primers''' | ||
| Line 91: | Line 91: | ||
PR-PhaR-NotI-for | PR-PhaR-NotI-for | ||
| - | 5-ATGCGGCCGCAGTGCCTTGTTGGGCATAGAATCAGGGCAGCGGCGCAGC-3 | + | <span style="color: Blue">5-ATGCGGCCGCAGTGCCTTGTTGGGCATAGAATCAGGGCAGCGGCGCAGC-3</span> |
PR-PhaR-NdeI-rev | PR-PhaR-NdeI-rev | ||
| - | 5-ACGAAGTTATCATATGTTATTACTTCTTGTCCGGCTGGTTGAACGGGAACGTCCCGAAC-3 | + | <span style="color: red">5-ACGAAGTTATCATATGTTATTACTTCTTGTCCGGCTGGTTGAACGGGAACGTCCCGAAC-3</span> |
LRLacILP-for-in | LRLacILP-for-in | ||
| - | 5-TGCTATACGAAGTTATAAAGAGGAGAAACTCGAGATGGTGAATGTGAAACCAGTAACGT-3 | + | <span style="color: Yellow">5-TGCTATACGAAGTTATAAAGAGGAGAAACTCGAGATGGTGAATGTGAAACCAGTAACGT-3</span> |
LRLacILP-NdeI-for-out | LRLacILP-NdeI-for-out | ||
| - | 5-ACAAGAAGTAATAACATATGATAACTTCGTATAATGTATGCTATACGAAGTTATAAAGA-3 | + | <span style="color: Green">5-ACAAGAAGTAATAACATATGATAACTTCGTATAATGTATGCTATACGAAGTTATAAAGA-3</span> |
LRLacILP-rev-in | LRLacILP-rev-in | ||
| - | 5-TACGAAGTTATCTACTGCCCGCTTTCCAGTCGGGAAACCT-3 | + | <span style="color: Orange">5-TACGAAGTTATCTACTGCCCGCTTTCCAGTCGGGAAACCT-3</span> |
LRLacILP-KpnI-rev-out | LRLacILP-KpnI-rev-out | ||
| - | 5-ATGGTACCATAACTTCGTATAGCATACATTATACGAAGTTATCTACTGCCCGCTT-3 | + | <span style="color: Brown">5-ATGGTACCATAACTTCGTATAGCATACATTATACGAAGTTATCTACTGCCCGCTT-3</span> |
'''(4) EGFP Primers''' | '''(4) EGFP Primers''' | ||
| Line 117: | Line 117: | ||
EGFP-for-KoZg-for | EGFP-for-KoZg-for | ||
| - | 5-ccatgggcagcaagggcgaggagctgttc-3 | + | <span style="color: red">5-ccatgggcagcaagggcgaggagctgttc-3</span> |
EGFP-rev-in | EGFP-rev-in | ||
| - | 5-AAAAAAAAACCCCGCCGAAGCGGGGTTTTTTTTTCTATCACTTGTACAGCTCGTC-3 | + | <span style="color: Green">5-AAAAAAAAACCCCGCCGAAGCGGGGTTTTTTTTTCTATCACTTGTACAGCTCGTC-3</span> |
EGFP-rev-out | EGFP-rev-out | ||
| - | 5-TTTTC GAGCTC GAATTC GGATCCAAAAAAAAACCCCGCCGAA-3 | + | <span style="color: Purple">5-TTTTC GAGCTC GAATTC GGATCCAAAAAAAAACCCCGCCGAA-3 |
| + | </span> | ||
== PCR == | == PCR == | ||
Revision as of 14:02, 29 October 2008
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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
PCR
Fusion PCR
Cut and Insert
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