Lab 208

Main purpose

• PCR to isolate Biobricks
• PCR to make the constitutive reference promoter
• colony PCR on Nir transformants

Who was in the lab

Kristian, Henrike

Procedure

PCR Biobrick Isolation

Used new primers to isolate Hello World constructs. Used HF buffer and tested three different concentrations of DMSO: 0%, 2% and 5%.

Primers: 56a, 56b

template: TAT2-1 (miniprep)

program:

PCR constitutive reference promoter

Got new primers for the constitutive reference promoter since the old reverse primer (52b2) had a mistake. Repeated reaction set-up and program from the 13.08. Used GC buffer, 5% DMSO and 2uL 50 mM MgCl2.

primers: 52a, 52bn

template: pZA21::RFP

program:

Colony PCR on Nir transformants

Used Q5 premix from NEB.

primers: 45a, 45b

program:

Results

Gel electrophoresis

• 1 kb ladder
• Sec2 Biobrick
• Sec2 Biobrick
• Sec2 neg
• col AMO 1
• col AMO 2
• col AMO 3
• col AMO 4
• col AMO 5
• col AMO 6
• col AMO 7
• col AMO 8
• col AMO 9
• col AMO 10
• 1 kb ladder

• 1 kb ladder
• col Nir
• constitutive reference promoter
• constitutive reference promoter
• Biobrick Isolation TAT2-1 negative
• Biobrick Isolation TAT2-1 2% DMSO
• Biobrick Isolation TAT2-1 5% DMSO
• Biobrick Isolation TAT2-1 w/o DMSO
• 1 kb ladder

Conclusion

The transformation of AMO into pZA21::ara was successful, all tested colonies have the insert.

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