Difference between revisions of "Part:BBa K2865002:Design"

 
Line 7: Line 7:
  
 
===Design Notes===
 
===Design Notes===
−
As we have already known, ITR regions are GC rich and have extensive secondary structures. The sequences of left and right ITRs are symmetry, making primers difficult to bind to the correct sites. Besides the complex structures, there's a Pst1 restriction site in both left and right ITR regions. Considering all the factors above, we decided to employ nested PCR to amplify ITRs. To tackle the problem of non-specific binding, two pairs of primers were desigend. The first was far from ITR core region and the second was approximal, with a base changed so as to remove Pst1 illegal site. The final product was a little bit longer than 145 bases, as the primers initiated from the outside of ITR region.
+
ITR regions are GC rich and have extensive secondary structures. The sequences of left and right ITRs are symmetry, making primers difficult to bind to the correct sites. Besides the complex structures, there's a Pst1 restriction site in both left and right ITR regions. Considering all the factors above, we decided to employ nested PCR to amplify ITRs. To tackle the problem of non-specific binding, two pairs of primers were desigend. The first was far from ITR core region and the second was approximal, with a base changed so as to remove Pst1 illegal site. The final product was a little bit longer than 145 bases, as the primers initiated from the outside of ITR region.
  
  

Revision as of 10:30, 30 September 2018


[AAV9]-Left-ITR


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Unknown
  • 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

ITR regions are GC rich and have extensive secondary structures. The sequences of left and right ITRs are symmetry, making primers difficult to bind to the correct sites. Besides the complex structures, there's a Pst1 restriction site in both left and right ITR regions. Considering all the factors above, we decided to employ nested PCR to amplify ITRs. To tackle the problem of non-specific binding, two pairs of primers were desigend. The first was far from ITR core region and the second was approximal, with a base changed so as to remove Pst1 illegal site. The final product was a little bit longer than 145 bases, as the primers initiated from the outside of ITR region.


Source

This part was cloned from an AAV-9 shuttle plasmid.

References