Team:HokkaidoU Japan/Notebook/September21
From 2010.igem.org
(Difference between revisions)
(5 intermediate revisions not shown) | |||
Line 10: | Line 10: | ||
For this I ordered oligo primers for sigma | For this I ordered oligo primers for sigma | ||
<br><br><br><br><br> | <br><br><br><br><br> | ||
- | == | + | == Arabinose induced expression of GFP == |
- | + | ||
*GFP-doubleterminator | *GFP-doubleterminator | ||
BBa_I13401 1-12K 875bp pSB1A2 | BBa_I13401 1-12K 875bp pSB1A2 | ||
- | * | + | *Arabinose promoter |
BBa_I0050 3-20B 1210bp pSB2K3 | BBa_I0050 3-20B 1210bp pSB2K3 | ||
*pSB1C3 2072bp | *pSB1C3 2072bp | ||
Line 21: | Line 20: | ||
<br> | <br> | ||
- | 1) | + | 1)PCR mix of Arabinose promoter and GFP, pSB1A3 |
- | {|style="text-align:center; | + | {|style="text-align:center; margin-left:100px;" class="protocol" |
!Reagent | !Reagent | ||
!Amount | !Amount | ||
Line 53: | Line 52: | ||
|style="border-top:1px solid #996;"|'''50 uL''' | |style="border-top:1px solid #996;"|'''50 uL''' | ||
|} | |} | ||
- | |||
2) | 2) | ||
- | + | * 94C 2min | |
- | + | * 98C 10sec | |
- | 68C 2min | + | * 68C 2min |
- | 35cycle | + | ** 35cycle |
- | 4C hold | + | * 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