Team:HokkaidoU Japan/Notebook/September21

From 2010.igem.org

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
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