Difference between revisions of "Part:BBa K2591013:Design"

(→‎Design Notes)
(→‎Design Notes)
 
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First, we use primers suffix_F(5'-TACTAGTAGCGGCCGCTGCAG-3') and prefix_R(5'-CTCTAGAAGCGGCCGCGAATTC-3') to do a reverse PCR with pSB1C3 backbone, obtaining the linearized backbone. Then we design the primers Prefix_LoxP_bGHpA_F(5'-GAATTCGCGGCCGCTTCTAGAGATAACTTCGTATAGCATACATTATACGAAGTTATTAGAGCTCGCTGATCAGCCTC-3') and Suffix_LoxP_bGHpA_R(5'-CTGCAGCGGCCGCTACTAGTAATAACTTCGTATAATGTATGCTATACGAAGTTATGAGCTCTCCCCAGCATGCCTGCTATTcTC-3') containing LoxP sequence, also, prefix and suffix sequence are added. Using these two primers and BGH polyA containing template to do PCR, we got the LSL part and then used Gibson Assembly to complete the plasmid.
 
First, we use primers suffix_F(5'-TACTAGTAGCGGCCGCTGCAG-3') and prefix_R(5'-CTCTAGAAGCGGCCGCGAATTC-3') to do a reverse PCR with pSB1C3 backbone, obtaining the linearized backbone. Then we design the primers Prefix_LoxP_bGHpA_F(5'-GAATTCGCGGCCGCTTCTAGAGATAACTTCGTATAGCATACATTATACGAAGTTATTAGAGCTCGCTGATCAGCCTC-3') and Suffix_LoxP_bGHpA_R(5'-CTGCAGCGGCCGCTACTAGTAATAACTTCGTATAATGTATGCTATACGAAGTTATGAGCTCTCCCCAGCATGCCTGCTATTcTC-3') containing LoxP sequence, also, prefix and suffix sequence are added. Using these two primers and BGH polyA containing template to do PCR, we got the LSL part and then used Gibson Assembly to complete the plasmid.
 +
In addition, we insert ScaI digestion site between LoxP sequence and BGH polyA, so that other teams can modify this part, inserting other components between two LoxP site.
  
 
===Source===
 
===Source===
  
−
First, we use primers suffix_F(5'-TACTAGTAGCGGCCGCTGCAG-3') and prefix_R(5'-CTCTAGAAGCGGCCGCGAATTC-3') to do a reverse PCR with pSB1C3 backbone, obtaining the linearized backbone. Then we design the primers Prefix_LoxP_bGHpA_F(5'-GAATTCGCGGCCGCTTCTAGAGATAACTTCGTATAGCATACATTATACGAAGTTATTAGAGCTCGCTGATCAGCCTC-3') and Suffix_LoxP_bGHpA_R(5'-CTGCAGCGGCCGCTACTAGTAATAACTTCGTATAATGTATGCTATACGAAGTTATGAGCTCTCCCCAGCATGCCTGCTATTcTC-3') containing LoxP sequence, also, prefix and suffix sequence are added. Using these two primers and BGH polyA containing template to do PCR, we got the LSL part and then used Gibson Assembly to complete the plasmid.
+
LoxP sequence is from synthesis, BGH polyA is from our host lab.
  
 
===References===
 
===References===
 +
 +
Jackson EL; Willis N; Mercer K; Bronson RT; Crowley D; Montoya R; Jacks T; Tuveson DA. 2001. Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras. Genes Dev 15(24):3243-8PubMed: 11751630MGI: J:73445

Latest revision as of 07:31, 7 October 2018


LoxP-BGH polyA-LoxP


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

First, we use primers suffix_F(5'-TACTAGTAGCGGCCGCTGCAG-3') and prefix_R(5'-CTCTAGAAGCGGCCGCGAATTC-3') to do a reverse PCR with pSB1C3 backbone, obtaining the linearized backbone. Then we design the primers Prefix_LoxP_bGHpA_F(5'-GAATTCGCGGCCGCTTCTAGAGATAACTTCGTATAGCATACATTATACGAAGTTATTAGAGCTCGCTGATCAGCCTC-3') and Suffix_LoxP_bGHpA_R(5'-CTGCAGCGGCCGCTACTAGTAATAACTTCGTATAATGTATGCTATACGAAGTTATGAGCTCTCCCCAGCATGCCTGCTATTcTC-3') containing LoxP sequence, also, prefix and suffix sequence are added. Using these two primers and BGH polyA containing template to do PCR, we got the LSL part and then used Gibson Assembly to complete the plasmid. In addition, we insert ScaI digestion site between LoxP sequence and BGH polyA, so that other teams can modify this part, inserting other components between two LoxP site.

Source

LoxP sequence is from synthesis, BGH polyA is from our host lab.

References

Jackson EL; Willis N; Mercer K; Bronson RT; Crowley D; Montoya R; Jacks T; Tuveson DA. 2001. Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras. Genes Dev 15(24):3243-8PubMed: 11751630MGI: J:73445