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 and Electrophoresis assay

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

PCR and Electrophoresis assay

10x Buffer KOD plus2mM 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

September 10

PCR and Electrophoresis assay

10x Buffer KOD plus2mM dNTPs25mM MgSO4F primerR primerDNA(CFP)KOD plusmilliQtotal
2.52.51.00.750.750.50.516.525

94℃ 2min, (94℃ 15sec, 48℃ 30sec)x30cycles, 68℃ 45sec

PCR and Electrophoresis assay

10x Buffer KOD plus2mM dNTPs25mM MgSO4F primerR primerDNA(lacI)KOD plusmilliQtotal
2.52.51.00.750.750.50.516.525

94℃ 2min, (94℃ 15sec, 51℃ 30sec)x30cycles, 68℃ 30sec

PCR and Electrophoresis assay

①PCR product of psB1K3 ②psB1K3 ③psB1C3 ④psB1K3 ⑤PCR product of CFP

10x Buffer KOD plus2mM dNTPs25mM MgSO4F primerR primerDNA(①-⑤)KOD plusmilliQtotal
2.52.51.00.750.750.50.516.525

94℃ 2min, (94℃ 15sec, 58℃ 30sec)x30cycles, 68℃ 2min20sec

PCR and Electrophoresis assay

①DT ②DT ③CFP ④CFP

10x Buffer KOD plus2mM dNTPs25mM MgSO4F primerR primerDNA(①-④)KOD plusmilliQtotal
2.52.51.00.750.750.50.516.525

94℃ 2min, (94℃ 15sec, 48℃ 30sec)x30cycles, 68℃ 1min

PCR and Electrophoresis assay

①DT ②DT ③DT

10x Buffer KOD plus2mM dNTPs25mM MgSO4F primerR primerDNA(lacI)KOD plusmilliQtotal
2.52.51.00.750.750.50.516.525

94℃ 2min, (94℃ 15sec, 50℃ 30sec)x30cycles, 68℃ 30sec

September 12

Golden Gate Assembly

psB1K3lacIGFP(2'-3)RFP(3'-5)DT10x NEB T4 Buffer100x BSABsaINEB T4 ligasemilliQtotal
1.80.11.00.60.11.50.151.01.08.1515
psB1K3lacIGFP(2'-5)DT10x NEB T4 Buffer100x BSABsaINEB T4 ligasemilliQtotal
1.80.11.00.11.50.151.01.08.7515
psB1K3lacIGFP(2'-3)RFP(3'-5)DT10x NEB T4 Buffer100x BSABsaINEB T4 ligasemilliQtotal
2.00.21.01.00.21.50.151.01.06.9515
psB1K3lacIGFP(2'-3)DT10x NEB T4 Buffer100x BSABsaINEB T4 ligasemilliQtotal
2.00.21.00.21.50.151.01.07.9515

September 12

Transformation

①lacI-GFP(2'-3)-RFP(3'-5)-DT ②lacI-GFP(2'-5)-DT ③lacI-GFP(2'-3)-RFP(3'-5)-DT ④lacI-GFP(2'-5)-DT

September 13

Transformation

①lacI-GFP(2'-3)-RFP(3'-5)-DT ②lacI-GFP(2'-5)-DT ③lacI-GFP(2'-3)-RFP(3'-5)-DT ④lacI-GFP(2'-5)-DT

September 14

Liquid Culture

①lacI-GFP(2'-3)-RFP(3'-5)-DT ②lacI-GFP(2'-5)-DT ③lacI-GFP(2'-3)-RFP(3'-5)-DT ④lacI-GFP(2'-5)-DT

September 15

Miniprep

①lacI-GFP(2'-3)-RFP(3'-5)-DT ②lacI-GFP(2'-5)-DT ③lacI-GFP(2'-3)-RFP(3'-5)-DT ④lacI-GFP(2'-5)-DT

①4.0 µg/ml ②17 µg/ml ③15 µg/ml ④8.0 µg/ml

Restriction Enzyme Digestion

①lacI-GFP(2'-3)-RFP(3'-5)-DT ②lacI-GFP(2'-5)-DT ③lacI-GFP(2'-3)-RFP(3'-5)-DT ④lacI-GFP(2'-5)-DT

DNA(①-④)10x NEB Bufer3BSAEcoR1MilliQTotal
920.50.5820

September 17

Electrophoresis assay

①lacI-GFP(2'-3)-RFP(3'-5)-DT ②lacI-GFP(2'-5)-DT ③lacI-GFP(2'-3)-RFP(3'-5)-DT ④lacI-GFP(2'-5)-DT

September 18

Restriction Enzyme Digestion

③lacI-GFP(2'-3)-RFP(3'-5)-DT ④lacI-GFP(2'-5)-DT

DNA(③,④)10x H BuferBSAEcoR1MilliQTotal
1530.50.51130

Colony PCR

①lacI-GFP(2'-3)-RFP(3'-5)-DT ②lacI-GFP(2'-5)-DT ③lacI-GFP(2'-3)-RFP(3'-5)-DT ④lacI-GFP(2'-5)-DT

2x Quick Tag Dye MixVF primerVR primerMiliQTotal
25112350

September 19

DpnI Restriction Enzyme Digestion of psB1K3

psB1K3DpnITotal
100.210.2

37℃ 1h incubate

Ligation of psB1K3

psB1K3Ligation HighMiliQTH KinaseTotal
257715

16.0℃　1.5h

September 20

Transformation of psB1K3

September 21

PCR of psB3C5 and Electrophoresis assay

10x Buffer KOD plus2mM dNTPs25mM MgSO4F primerR primerDNA(psB3C5)KOD plusmilliQtotal
2.52.51.00.750.750.50.516.525

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

PCR and Electrophoresis assay

①psB3C5 ②psB1K3

10x Buffer KOD plus2mM dNTPs25mM MgSO4F primerR primerDNA(①,②)KOD plusmilliQtotal
5.05.02.01.51.51.01.03350

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

PCR of DT and Electrophoresis assay

10x Buffer KOD plus2mM dNTPs25mM MgSO4F primerR primerDNA(DT)KOD plusmilliQtotal
5.05.02.01.51.51.01.03350

94℃ 2min, (94℃ 15sec, 50℃ 30sec, 68℃ 30sec)x30cycles

September 22

Colony PCR (Colony plated in September 20)

10x Buffer KOD plus2mM dNTPs25mM MgSO4F primerR primerDNAKOD plusmilliQtotal
5.05.02.01.51.51.01.03350

94℃ 2min, (94℃ 15sec, 58℃ 30sec, 68℃ 2min20sec)x25cycles

PCR of Primers and Electrophoresis assay

①J23106(1'-2) ②J23106(2'-3) ③J23106(3'-4) ④J23106(4'-5) ⑤J23106(3'-5) ⑥J23106(2'-5) ⑦J23106(1'-5) ⑧araC(1'-6) ⑨araC(5'-6)

10x Buffer KOD plus2mM dNTPs25mM MgSO4F primerR primerDNAKOD plusmilliQtotal
2.52.51.00.750.750.50.516.525

94℃ 2min, (94℃ 15sec, 56℃ 30sec)x30cycles, 68℃ 20sec

①J23106(1'-2) 60µg/ml ②J23106(2'-3) 63µg/ml ③J23106(3'-4) 48µg/ml ④J23106(4'-5) 60µg/ml ⑤J23106(3'-5) 62µg/ml ⑥J23106(2'-5) 40µg/ml ⑦J23106(1'-5) 29µg/ml ⑧araC(1'-6) 60µg/ml ⑨araC(5'-6) 62µg/ml

Ligation

psB3C5①J23106(1'-2)②J23106(2'-3)③J23106(3'-4)④J23106(4'-5)⑨araC(5'-6)GFPDT10x NEB T4 Buffer100x BSABsaINEB T4 ligasemilliQtotal
2.02.02.02.52.02.03.03.01.50.151.01.07.8530
psB3C5①J23106(1'-2)②J23106(2'-3)⑤J23106(3'-5)⑨araC(5'-6)GFPDT10x NEB T4 Buffer100x BSABsaINEB T4 ligasemilliQtotal
2.02.02.02.52.03.03.01.50.151.01.07.8530
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