Team:HokkaidoU Japan/Notebook/September21

From 2010.igem.org

(Difference between revisions)
 
(10 intermediate revisions not shown)
Line 1: Line 1:
{{Template:HokkaidoU_Japan}}
{{Template:HokkaidoU_Japan}}
<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/September20|September 20]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/September22|September 22]]</div></div>
<div class="linkbar"><div class="prev">[[Team:HokkaidoU_Japan/Notebook/September20|September 20]]</div>[[Team:HokkaidoU_Japan/Notebook|Notebook]]<div class="next">[[Team:HokkaidoU_Japan/Notebook/September22|September 22]]</div></div>
 +
 +
 +
== Annealing of RBS made from oligos ==
 +
 +
[[Image:HokkaidoU_Japan_RBS_B0034.png‎|200px|right|frame| Construction of RBS (BBa_B0034) from oligos. Already precut!]]
 +
Annealing of RBS (BBa_B0034) made from oligos
 +
 +
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

Notebook


Annealing of RBS made from oligos

Construction of RBS (BBa_B0034) from oligos. Already precut!

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

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
Retrieved from "http://2010.igem.org/Team:HokkaidoU_Japan/Notebook/September21"