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Team:Kyoto/GoldenGateAssembly/Notebook
From 2012.igem.org
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==September 6== | ==September 6== | ||
====PCR==== | ====PCR==== | ||
| - | + | ①psB1K3 | |
②lacI | ②lacI | ||
③GFP | ③GFP | ||
| Line 27: | Line 27: | ||
==September 7== | ==September 7== | ||
====Electrophoresis assay==== | ====Electrophoresis assay==== | ||
| - | + | ①psB1K3 | |
| + | ②lacI | ||
| + | ③GFP | ||
| + | ④GFP | ||
| + | ⑤RFP | ||
| + | ⑥RFP | ||
| + | ⑦CFP | ||
| + | ⑧DT | ||
| + | ====PCR of psB1K3==== | ||
| + | {|class="wikitable" | ||
| + | !10x Buffer!!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 | ||
</div> | </div> | ||
{{Kyoto/footer}} | {{Kyoto/footer}} | ||
Revision as of 07:47, 20 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
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
Electrophoresis assay
①psB1K3 ②lacI ③GFP ④GFP ⑤RFP ⑥RFP ⑦CFP ⑧DT
PCR of psB1K3
| 10x Buffer | 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
