Team:HokkaidoU Japan/Notebook/September21
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For this I ordered oligo primers for sigma | For this I ordered oligo primers for sigma | ||
+ | <br><br><br><br><br> | ||
+ | == Arabinose induced expression of GFP == | ||
+ | *GFP-doubleterminator | ||
+ | BBa_I13401 1-12K 875bp pSB1A2 | ||
+ | *Arabinose promoter | ||
+ | BBa_I0050 3-20B 1210bp pSB2K3 | ||
+ | *pSB1C3 2072bp | ||
+ | *pSB1A3 2157bp | ||
+ | |||
+ | <br> | ||
+ | |||
+ | 1)PCR mix of Arabinose promoter and GFP, pSB1A3 | ||
+ | {|style="text-align:center; margin-left:100px;" class="protocol" | ||
+ | !Reagent | ||
+ | !Amount | ||
+ | |- | ||
+ | |Dw | ||
+ | |33 uL | ||
+ | |- | ||
+ | |10×PCR buffur | ||
+ | |5 | ||
+ | |- | ||
+ | |2mM 4dNTP | ||
+ | |5 | ||
+ | |- | ||
+ | |MgSO4 | ||
+ | |3 | ||
+ | |- | ||
+ | |Ex-F | ||
+ | |1 | ||
+ | |- | ||
+ | |Ps-R | ||
+ | |1 | ||
+ | |- | ||
+ | |KOD neo plus | ||
+ | |1 | ||
+ | |- | ||
+ | |DNA | ||
+ | |1 | ||
+ | |- | ||
+ | |style="border-top:1px solid #996;"|'''Total''' | ||
+ | |style="border-top:1px solid #996;"|'''50 uL''' | ||
+ | |} | ||
+ | |||
+ | 2) | ||
+ | * 94C 2min | ||
+ | * 98C 10sec | ||
+ | * 68C 2min | ||
+ | ** 35cycle | ||
+ | * 4C hold | ||
+ | |||
+ | 3)After PCR, Electrophored and extracted samples | ||
+ | |||
+ | 4)Estimated the concentration of the samples | ||
+ | |||
+ | <div style="margin-left:100px;"> | ||
+ | [[Image:HokkaidoU_Japan_20100921a.JPG|200px|thumb|left|泳動結果]] | ||
+ | |||
+ | |||
+ | from left to right | ||
+ | |||
+ | 1.λ/Hind III, EcoR I | ||
+ | |||
+ | 2.Arabinose promoter | ||
+ | |||
+ | 3.GFP+double terminator | ||
+ | |||
+ | 4.pSB1A3 | ||
+ | |||
+ | 5.RFP reporter | ||
+ | |||
+ | 6.RFP? | ||
+ | |||
+ | 7.Heat shock promoter</div> |
Latest revision as of 02:35, 28 October 2010
Annealing of RBS made from oligos
Annealing of RBS (BBa_B0034) made from oligos
For this I ordered oligo primers for sigma
Arabinose induced expression of GFP
- GFP-doubleterminator
BBa_I13401 1-12K 875bp pSB1A2
- Arabinose promoter
BBa_I0050 3-20B 1210bp pSB2K3
- pSB1C3 2072bp
- pSB1A3 2157bp
1)PCR mix of Arabinose promoter and GFP, pSB1A3
Reagent | Amount |
---|---|
Dw | 33 uL |
10×PCR buffur | 5 |
2mM 4dNTP | 5 |
MgSO4 | 3 |
Ex-F | 1 |
Ps-R | 1 |
KOD neo plus | 1 |
DNA | 1 |
Total | 50 uL |
2)
- 94C 2min
- 98C 10sec
- 68C 2min
- 35cycle
- 4C hold
3)After PCR, Electrophored and extracted samples
4)Estimated the concentration of the samples