Difference between revisions of "Part:BBa K187293:Design"

 
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−
 
 
__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K187293 short</partinfo>
 
<partinfo>BBa_K187293 short</partinfo>
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===Design Notes===
 
===Design Notes===
 +
Design considerations:
 +
 
This primer produced a product of the predicted size using the following reaction conditions:
 
This primer produced a product of the predicted size using the following reaction conditions:
 
:
 
:
 
Water: 17.05uL
 
Water: 17.05uL
 +
 
10x pfu buffer: 2.5uL
 
10x pfu buffer: 2.5uL
 +
 
dNTPs (2mM): 2.5uL
 
dNTPs (2mM): 2.5uL
 +
 
DMSO: 1.2uL
 
DMSO: 1.2uL
 +
 
MG1655 Genomic DNA: 0.5uL
 
MG1655 Genomic DNA: 0.5uL
 +
 
Forward primer (10uM): 0.5uL
 
Forward primer (10uM): 0.5uL
 +
 
Reverse primer (10uM): 0.5uL
 
Reverse primer (10uM): 0.5uL
 +
 
Pfu polymerase: 0.25uL
 
Pfu polymerase: 0.25uL
  
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Thermocycling conditions:
 
Thermocycling conditions:
 +
 
95C, 3min
 
95C, 3min
 +
 
95C, 30s
 
95C, 30s
 +
 
56C, 30s
 
56C, 30s
 +
 
72C, 3min
 
72C, 3min
 +
 
29 cycles to step 2
 
29 cycles to step 2
 +
 
72C 2min
 
72C 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:
 
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.  
 
Find the shortest possible sequence, reducing the cost to produce the primer.  
 +
 
Produce the highest score value possible.  
 
Produce the highest score value possible.  
 +
 
Produce the closest Tm's possible  
 
Produce the closest Tm's possible  
−
Produce hairpins with dG values >-5  
+
 
 +
Produce hairpins with dG values >-5
 +
 
 
Produce dimers with dG values >-10  
 
Produce dimers with dG values >-10  
 +
 
The following are Vector NTI statistics for this primer:
 
The following are Vector NTI statistics for this primer:
−
dG Dimer (kcal/mol): -8.4
+
 
−
dG Hairpin (kcal/mol): -9.1
+
dG Dimer (kcal/mol): (from the spreadsheet)
 +
 
 +
dG Hairpin (kcal/mol): (from the spreadsheet)
  
  

Revision as of 22:16, 20 October 2009

mnmA ORF, Forward Primer


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


Design Notes

Design considerations:

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:

95C, 3min

95C, 30s

56C, 30s

72C, 3min

29 cycles to step 2

72C 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): (from the spreadsheet)

dG Hairpin (kcal/mol): (from the spreadsheet)



Source

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


References