Molecular Cloning

From 2008.igem.org

(Difference between revisions)
(PCR)
(Fragment Preparation)
Line 189: Line 189:
SRRz: PCR with primer SRRZ-NcoI-for and SRRZ-rev-in;
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;
+
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;
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;
+
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;
PpPhaP: PCR with primer PP-PhaP-NotI-for and PP-PhaP-HindIII-rev;
Line 199: Line 199:
LacI(LVA): PCR with primer LacILVA-HindIII-for and LacILVA-rev-in;
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;
+
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
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;
+
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;
PrPhaR: PCR with primer PR-PhaR-NotI-for and PR-PhaR-NdeI-rev;
Line 209: Line 209:
LoxPLacILoxP: PCR with primer LRLacILP-for-in and LRLacILP-rev-in
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.
+
Purify the product and run PCR with primer LRLacILP-NdeI-for-out and primer LRLacILP-KpnI-rev-out.
== Fusion PCR==
== Fusion PCR==

Revision as of 14:07, 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

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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