Primer

Part:BBa_K187198:Design

Designed by: Anh Dao   Group: iGEM09_Alberta   (2009-10-19)


pyrG, ORF, reverse primer


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 9
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 9
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 9
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

This primer produced a product of the predicted size using the following reaction conditions:

Water: 17.05uL

10x pfu buffer: 2.5uL

dNTPs (2mM): 2.5uL

DMSO: 1.2uL

MG1655 Genomic DNA: 0.5uL

Forward primer (10uM): 0.5uL

Reverse primer (10uM): 0.5uL

Pfu polymerase: 0.25uL

Total reaction volume: 25uL

Thermocycling conditions:

95oC, 3min

95oC, 30s

56oC, 30s

72oC, 3min

29 cycles to step 2

72oC 2min

All Biobytes essential gene primers were produced using Vector NTI. Each primer's thermodynamic properties were examined using Vector NTI's hairpin and dimerzation programs. Primers were optimized to fit as many of the following criteria as possible: 

  * Find the shortest possible sequence, reducing the cost to produce the primer.

  * Produce the highest score value possible.

  * Produce the closest Tm's possible

  * Produce hairpins with dG values >-5

  * Produce dimers with dG values >-10

The following are Vector NTI statistics for this primer:

dG Dimer (kcal/mol): -4.7

dG Hairpin (kcal/mol): -0.3


Source

Oligonucleotides were synthesized. Primers were tested using MG1655 E.coli genomic DNA as template.

References