Difference between revisions of "Part:BBa K187291:Design"
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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 | ||
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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: | ||
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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: | ||
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dG Dimer (kcal/mol): -2.3 | dG Dimer (kcal/mol): -2.3 | ||
| + | |||
dG Hairpin (kcal/mol): -1.4 | dG Hairpin (kcal/mol): -1.4 | ||
Latest revision as of 22:19, 20 October 2009
metK ORF forward primer
- 10INCOMPATIBLE WITH RFC[10]Illegal XbaI site found at 9
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal XbaI site found at 9
- 25INCOMPATIBLE WITH RFC[25]Illegal XbaI site found at 9
- 1000COMPATIBLE 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:
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): -2.3
dG Hairpin (kcal/mol): -1.4
Source
Oligonucleotides were synthesized. Primers were tested using MG1655 E.coli genomic DNA as template