User:Cesar A. Villavicencio/Notebook/Tyrosinase Extraction/2012/05/01

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Primer Order and Design

6 Primers were designed for tyrosinase extraction from Bacillus megaterium QMB1551 for the extraction and in situ mutation of the gene. The primers designed were the following


Primers for Tyrosinase Extraction

TyrFWD

5'-GAATTCGCCCCTTGCTTCTTT-3'

Length=21 A=2 T=9 C=7 G=3 CG=47.62%

Linguistic Complexity=82%

Primer's PCR Efficiency=85%

Tm=56.4°C (Allawi's thermodynamics parameters)**

Tm=56.4°C (Tm = 77.1 + 11.7Log[K+] + 0.41(GC%) - 528/L)

Extinction coefficient = 178200 L/(mol•cm)

Molecular weight = 6314.13 g/mol


TyrREV

5'-CTGCTTTTGATTGCATACCG-3'

Length=20 A=3 T=8 C=5 G=4 CG=45%

Linguistic Complexity=85%

Primer's PCR Efficiency=85%

Tm=52.9°C (Allawi's thermodynamics parameters)**

Tm=54.3°C (Tm = 77.1 + 11.7Log[K+] + 0.41(GC%) - 528/L)

Extinction coefficient = 179100 L/(mol•cm)

Molecular weight = 6073.98 g/mol



Primers for in situ mutation

MutTyrFWD

5'-gacgcacagctccaagatccctcacag-3'

Length=27 A=8.0 G=5.0 T=3.0 C=11.0 CG=59.3%

Linguistic complexity = 78%

Primer's PCR efficiency = 88%


Tm = 64.1°C (Allawi's thermodynamics parameters)

Tm = 69.1°C (Sugimoto's thermodynamics parameters)

Tm = 52.1°C (SantaLucia's thermodynamics parameters)

Tm = 64.6°C (Tm = 75.1 + 11.7Log[K+] + 0.41(GC%) - 528/L)

Tm = 64.3°C (Tm = 64.9 + 41(nG + nC - 16.4)/L)


MutTyrREV

5'-ctgtgagggatcttggagctgtgcgtc-3'

Length=27 A=3.0 G=11.0 T=8.0 C=5.0 CG=59.3%

Linguistic complexity = 78%

Primer's PCR efficiency = 93%

Tm = 64.1°C (Allawi's thermodynamics parameters)

Tm = 69.1°C (Sugimoto's thermodynamics parameters)

Tm = 57.6°C (SantaLucia's thermodynamics parameters)

Tm = 64.6°C (Tm = 75.1 + 11.7Log[K+] + 0.41(GC%) - 528/L)

Tm = 64.3°C (Tm = 64.9 + 41(nG + nC - 16.4)/L)



Primers for BioBrick adaptation

BMTyrFWD

5'-gaattcgcggccgcttctagaggaaagaggttaaaaaatgagtaacaagtac-3'

Length=52 A=20.0 G=14.0 T=10.0 C=8.0 CG=42.3%

Linguistic complexity = 82%

Primer's PCR efficiency = 39%

Tm = 65.9°C (Allawi's thermodynamics parameters)

Tm = 73.4°C (Sugimoto's thermodynamics parameters)

Tm = 62.2°C (SantaLucia's thermodynamics parameters)

Tm = 67.1°C (Tm = 75.1 + 11.7Log[K+] + 0.41(GC%) - 528/L)

Tm = 69.3°C (Tm = 64.9 + 41(nG + nC - 16.4)/L)


BMTyrREV

5'-ctgcagcggccgctactagtaaacgggttatgatgaacgtt-3'

Length=41 A=10.0 G=12.0 T=10.0 C=9.0 CG=51.2%

Linguistic complexity = 86%

Primer's PCR efficiency = 17%


Tm = 68.1°C (Allawi's thermodynamics parameters)

Tm = 74.4°C (Sugimoto's thermodynamics parameters)

Tm = 62.9°C (SantaLucia's thermodynamics parameters)

Tm = 68.0°C (Tm = 75.1 + 11.7Log[K+] + 0.41(GC%) - 528/L)

Tm = 69.5°C (Tm = 64.9 + 41(nG + nC - 16.4)/L)




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