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Team:Kyoto/GoldenGateAssembly/Notebook
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!10x Buffer KOD plus!!2mM dNTPs!!25mM MgSO4!!F primer!!R primer!!DNA(psB3C5)!!KOD plus!!milliQ!!total | !10x Buffer KOD plus!!2mM dNTPs!!25mM MgSO4!!F primer!!R primer!!DNA(psB3C5)!!KOD plus!!milliQ!!total | ||
Revision as of 07:39, 25 September 2012
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 Taq | primer F | primer R | DNA(①-⑧) | MilliQ | Total |
|---|---|---|---|---|---|
| 12.5 | 0.5 | 0.5 | 1 | 10.5 | 25 |
94℃ 2min, (94℃ 30sec, 50℃ 30sec)x25cycles, 68℃ 50sec
September 7
PCR and Electrophoresis assay
| 10x Buffer KOD plus | 2mM dNTPs | 25mM MgSO4 | F primer | R primer | DNA(psB1K3) | KOD plus | milliQ | total |
|---|---|---|---|---|---|---|---|---|
| 2.5 | 2.5 | 1.0 | 0.75 | 0.75 | 0.5 | 0.5 | 16.5 | 25 |
94℃ 2min, (94℃ 15sec, 58℃ 30sec)x30cycles, 68℃ 2min
September 10
PCR and Electrophoresis assay
| 10x Buffer KOD plus | 2mM dNTPs | 25mM MgSO4 | F primer | R primer | DNA(CFP) | KOD plus | milliQ | total |
|---|---|---|---|---|---|---|---|---|
| 2.5 | 2.5 | 1.0 | 0.75 | 0.75 | 0.5 | 0.5 | 16.5 | 25 |
94℃ 2min, (94℃ 15sec, 48℃ 30sec)x30cycles, 68℃ 45sec
PCR and Electrophoresis assay
| 10x Buffer KOD plus | 2mM dNTPs | 25mM MgSO4 | F primer | R primer | DNA(lacI) | KOD plus | milliQ | total |
|---|---|---|---|---|---|---|---|---|
| 2.5 | 2.5 | 1.0 | 0.75 | 0.75 | 0.5 | 0.5 | 16.5 | 25 |
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 plus | 2mM dNTPs | 25mM MgSO4 | F primer | R primer | DNA(①-⑤) | KOD plus | milliQ | total |
|---|---|---|---|---|---|---|---|---|
| 2.5 | 2.5 | 1.0 | 0.75 | 0.75 | 0.5 | 0.5 | 16.5 | 25 |
94℃ 2min, (94℃ 15sec, 58℃ 30sec)x30cycles, 68℃ 2min20sec
PCR and Electrophoresis assay
①DT ②DT ③CFP ④CFP
| 10x Buffer KOD plus | 2mM dNTPs | 25mM MgSO4 | F primer | R primer | DNA(①-④) | KOD plus | milliQ | total |
|---|---|---|---|---|---|---|---|---|
| 2.5 | 2.5 | 1.0 | 0.75 | 0.75 | 0.5 | 0.5 | 16.5 | 25 |
94℃ 2min, (94℃ 15sec, 48℃ 30sec)x30cycles, 68℃ 1min
PCR and Electrophoresis assay
①DT ②DT ③DT
| 10x Buffer KOD plus | 2mM dNTPs | 25mM MgSO4 | F primer | R primer | DNA(lacI) | KOD plus | milliQ | total |
|---|---|---|---|---|---|---|---|---|
| 2.5 | 2.5 | 1.0 | 0.75 | 0.75 | 0.5 | 0.5 | 16.5 | 25 |
94℃ 2min, (94℃ 15sec, 50℃ 30sec)x30cycles, 68℃ 30sec
September 12
Golden Gate Assembly
| psB1K3 | lacI | GFP(2'-3) | RFP(3'-5) | DT | 10x NEB T4 Buffer | 100x BSA | BsaI | NEB T4 ligase | milliQ | total |
|---|---|---|---|---|---|---|---|---|---|---|
| 1.8 | 0.1 | 1.0 | 0.6 | 0.1 | 1.5 | 0.15 | 1.0 | 1.0 | 8.15 | 15 |
| psB1K3 | lacI | GFP(2'-5) | DT | 10x NEB T4 Buffer | 100x BSA | BsaI | NEB T4 ligase | milliQ | total |
|---|---|---|---|---|---|---|---|---|---|
| 1.8 | 0.1 | 1.0 | 0.1 | 1.5 | 0.15 | 1.0 | 1.0 | 8.75 | 15 |
| psB1K3 | lacI | GFP(2'-3) | RFP(3'-5) | DT | 10x NEB T4 Buffer | 100x BSA | BsaI | NEB T4 ligase | milliQ | total |
|---|---|---|---|---|---|---|---|---|---|---|
| 2.0 | 0.2 | 1.0 | 1.0 | 0.2 | 1.5 | 0.15 | 1.0 | 1.0 | 6.95 | 15 |
| psB1K3 | lacI | GFP(2'-3) | DT | 10x NEB T4 Buffer | 100x BSA | BsaI | NEB T4 ligase | milliQ | total |
|---|---|---|---|---|---|---|---|---|---|
| 2.0 | 0.2 | 1.0 | 0.2 | 1.5 | 0.15 | 1.0 | 1.0 | 7.95 | 15 |
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 Bufer3 | BSA | EcoR1 | MilliQ | Total |
|---|---|---|---|---|---|
| 9 | 2 | 0.5 | 0.5 | 8 | 20 |
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 Bufer | BSA | EcoR1 | MilliQ | Total |
|---|---|---|---|---|---|
| 15 | 3 | 0.5 | 0.5 | 11 | 30 |
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 Mix | VF primer | VR primer | MiliQ | Total |
|---|---|---|---|---|
| 25 | 1 | 1 | 23 | 50 |
September 19
DpnI Restriction Enzyme Digestion of psB1K3
| psB1K3 | DpnI | Total |
|---|---|---|
| 10 | 0.2 | 10.2 |
37℃ 1h incubate
Ligation of psB1K3
| psB1K3 | Ligation High | MiliQ | TH Kinase | Total |
|---|---|---|---|---|
| 2 | 5 | 7 | 7 | 15 |
16.0℃ 1.5h
September 20
Transformation of psB1K3
September 21
PCR of psB3C5 and
| 10x Buffer KOD plus | 2mM dNTPs | 25mM MgSO4 | F primer | R primer | DNA(psB3C5) | KOD plus | milliQ | total |
|---|---|---|---|---|---|---|---|---|
| 2.5 | 2.5 | 1.0 | 0.75 | 0.75 | 0.5 | 0.5 | 16.5 | 25 |
94℃ 2min, (94℃ 15sec, 58℃ 30sec)x30cycles, 68℃ 2min
