Team:Kyoto/GoldenGateAssembly/Notebook

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Contents

Golden Gate Assembly Notebook

August 10

Golden Gate Assembly method This method helps us to constract some genes quickly and we can design the order of constractions. We are trying to construct a gene cycle composed of 6 genes( pSB1A3, lacI, GFP, RFP, GFP, DT). After this experiment is confirmed to work, we will so some experiment to check how the numbers of promorters influences the strength of transcription.

September 6

PCR

①psB1K3 ②lacI ③GFP ④GFP ⑤RFP ⑥RFP ⑦CFP ⑧DT

Quick Taqprimer Fprimer RDNA(①-⑧)MilliQTotal
12.50.50.5110.525

94℃ 2min, (94℃ 30sec, 50℃ 30sec)x25cycles, 68℃ 50sec

September 7

Electrophoresis assay

①psB1K3 ②lacI ③GFP ④GFP ⑤RFP ⑥RFP ⑦CFP ⑧DT

PCR of psB1K3

10x Buffer2mM dNTPs25mM MgSO4F primerR primerDNA(psB1K3)KOD plusmilliQtotal
2.52.51.00.750.750.50.516.525

94℃ 2min, (94℃ 15sec, 58℃ 30sec)x30cycles, 68℃ 2min