IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/09/16

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9/16/10

All overnights were white!!!

12:30 PM

Miniprep Promega kit

J23100B0034ArsRSDM2B0015pSB1T3

colony 1: 76.4 ng/μL

colony 2: 75.9 ng/μL

colony 3: 6.5 ng/μL

colony 4: 50.2 ng/μL

diluted colonies 1,2, and 4 to 20 ng/μL

1:30 PM

Verification PCR

Ingredient Amount Mastermix (5 reactions)
10X Buffer 2 μL 10 μL
10 mM dNTPs .4 μL 2 μL
Template 1 μL (20 ng) added individually to each reaction
F Primer .4 μL 2 μL
R Primer .4 μL 2 μL
Polymerase .2 μL 1 μL
H2O 15.6 μL 78 μL

Reaction for J23100B0034ArsRSDM2B0015pSB1T3 3

Ingredient Amount
10X Buffer 2 μL
10 mM dNTPs .4 μL
Template 3.07 μL (20ng)
F Primer .4 μL
R Primer .4 μL
Polymerase .2 μL
H2O 13.53 μL

Samples:

  1. J23100B0034ArsRSDM2B0015pSB1T3 1
  2. J23100B0034ArsRSDM2B0015pSB1T3 2
  3. J23100B0034ArsRSDM2B0015pSB1T3 3
  4. J23100B0034ArsRSDM2B0015pSB1T3 4
  5. negative control (H2O)

Primers (10 micromolar):

  • VF2
  • VR

Program:

95°C 3 min
95°C 30 sec
55°C 30 sec
72°C 1 min
go to step 2 30X
72°C 3 min
hold at 4°C

5:20 PM

Gel

TAE, 1%, 175V

Lane Sample Amount
1 1 Kb ladder 1 μL dye
2 J23100B0034ArsRSDM2B0015pSB1T3 1 1 μL dye, 5 μL sample
3 J23100B0034ArsRSDM2B0015pSB1T3 2 1 μL dye, 5 μL sample
4 J23100B0034ArsRSDM2B0015pSB1T3 3 1 μL dye, 5 μL sample
5 J23100B0034ArsRSDM2B0015pSB1T3 4 1 μL dye, 5 μL sample
6 negative control 1 μL dye, 5 μL sample

Image: UIUC_9.16.10_arsrj23100_verification.jpg‎


Made glycerol stock of J23100B0034ArsRSDM2B0015pSB13 colony 1, ISB237



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