Difference between revisions of "Part:BBa K192000:Design"
(→‎Helpful Tips)
Line 14: Line 14:
 
**5'CTT CTA GAT GGA ATT TCT GAA AAG ATC
 
**5'CTT CTA GAT GGA ATT TCT GAA AAG ATC
  
−
**Backward Primer  
+
*Backward Primer  
−
*** 5'CTA CTA GTA TCA TCA GAA CTT TAG AAG AAT CA
+
** 5'CTA CTA GTA TCA TCA GAA CTT TAG AAG AAT CA
  
 
*TD-PCR amplification temperatures:
 
*TD-PCR amplification temperatures:

Revision as of 19:22, 21 October 2009

Encapsulin


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 74
    Illegal XhoI site found at 274
    Illegal XhoI site found at 568
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Sequence was PCR'd from T. Maritima. An additional tga stop site was added.


Helpful Tips

TD-PCR

  • Forward Primer
    • 5'CTT CTA GAT GGA ATT TCT GAA AAG ATC
  • Backward Primer
    • 5'CTA CTA GTA TCA TCA GAA CTT TAG AAG AAT CA
  • TD-PCR amplification temperatures:

Notes

  • Primers
    • The primers used and noted above contain a SpeI site downstream and a Xbal site upstream for "easy" ligation into a biobrick plasmid.
  • Polymerase that was used was KapaHiFi (Engineered variant of of Taq)
    • Required to increase the [] of MgCl2.
    • After amplification at the specified TD-PCR program there was no unexpected bands


Suggestions

  • Primers
    • For better enzymatic digestion of SpeI and Xbal it was suggested to extend the primers beyond the recognition sequence by a few base pairs to allow something for the enzymes get a "grip-on"
      • As long as 3' end of primer is complementary there should be no issues with primer annealing.
    • TD-PCR was done to ensure we had product to extract and subject to another round of PCR to optimize , however due to lack of non-specific products the PCR protocol was never optimized.

Source

Thermotoga Maritima

References