Template:Kyoto/Notebook/GoldenGate

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Contents

Kyoto GoldenGateAssemblyNotebook.png

September 6

PCR and Electrophoresis assay

Epa001.jpg

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 of psB1K3

Epa002.jpg
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 of CFP

Epa002.jpg
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 of lacI

Epa003.jpg
10x Buffer KOD plus2mM dNTPs25mM MgSO4F primerR primerDNA(lacI)KOD plusmilliQtotal
2.52.51.00.750.750.50.516.525

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

PCR

①PCR product of psB1K3 ②psB1K3 ③psB1C3 ④psB1C3 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

September 11

PCR and Electrophoresis assay

Epa004.jpg

①DT ②PCR product of DT ③PCR product of CFP ④CFP(2011 plate)

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

Epa005.jpg

①DT 92.1ng/µl ②DT 28.0ng/µl ③DT

10x Buffer KOD plus2mM dNTPs25mM MgSO4F primerR primerDNA(①-③)KOD plusmilliQtotal
2.52.51.00.750.750.50.516.525

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

September 12

Purification of PCR product

psB1K3(9/10④) 55µg/ml lacI(9/10) 90µg/ml GFP(9/6) 42µg/ml RFP(9/6) 71µg/ml DT(9/11) 81µg/ml GFP 37µg/ml RFP 57µg/ml

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

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

Epa006.jpg

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

Epa007.jpg

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

Epa008.jpg
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

Epa009.jpg

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

Epa011.jpg
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)

Epa012.jpg
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

Epa013.jpg

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

4 promoters

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

3 promoters

psB3C5①J23106 (1'-2)②J23106 (2'-3)⑤J23106 (3'-5)⑨araC (5'-6)GFPDT10x NEB T4 Buffer100x BSABsaINEB T4 ligasemilliQtotal
2.02.02.02.52.03.03.01.50.151.01.09.8530

2 promoters

psB3C5①J23106(1'-2)⑥J23106(2'-5)⑨araC(5'-6)GFPDT10x NEB T4 Buffer100x BSABsaINEB T4 ligasemilliQtotal
2.02.02.52.03.03.01.50.151.011.8530

1 promoter

psB3C5⑦J23106(1'-5)⑨araC(5'-6)GFPDT10x NEB T4 Buffer100x BSABsaINEB T4 ligasemilliQtotal
2.02.52.03.03.01.50.151.01.013.8530

0 promoter

psB3C5⑨araC(5'-6)GFPDT10x NEB T4 Buffer100x BSABsaINEB T4 ligasemilliQtotal
2.02.03.03.01.50.151.01.016.3530

September 23

Transformation of product of ligation(9/22)

September 24

Transformation of product of ligation(9/22)

September 25

Liquid Culture

colony1,2,3 of plate2,3

Ligation

4 promoters

psB3C5① J23106 (1'-2)② J23106 (2'-3)③ J23106 (3'-4)④ J23106 (4'-5)⑨ araC (5'-6)GFPDT10x NEB T4 Buffer100x BSABsaINEB T4 ligasemilliQtotal
2.00.20.30.20.20.21.00.21.50.151.01.07.0515

3 promoters

psB3C5①J23106 (1'-2)②J23106 (2'-3)⑤J23106 (3'-5)⑨araC (5'-6)GFPDT10x NEB T4 Buffer100x BSABsaINEB T4 ligasemilliQtotal
2.00.20.30.20.21.00.21.50.151.01.07.2515

2 promoters

psB3C5①J23106 (1'-2)⑥J23106 (2'-5)⑨araC (5'-6)GFPDT10x NEB T4 Buffer100x BSABsaINEB T4 ligasemilliQtotal
2.00.20.30.21.00.21.50.151.01.07.4515

1 promoter

psB3C5⑦J23106 (1'-5)⑨araC (5'-6)GFPDT10x NEB T4 Buffer100x BSABsaINEB T4 ligasemilliQtotal
2.00.60.21.00.21.50.151.01.07.3515

0 promoter

psB3C5⑨araC (5'-6)GFPDT10x NEB T4 Buffer100x BSABsaINEB T4 ligasemilliQtotal
2.00.21.00.21.50.151.01.07.9515

(37℃ 3min, 16℃ 4min)x25cycles,

Transformation of product of ligation(9/25)