IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/09/23

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9.23.10

12:05 PM

PCR Verification

Ingredient Amount Mastermix (6 reactions)
10X Buffer 2 μL 12 μL
10 mM dNTPs .4 μL 2.4 μL
Template 1 μL (20ng) added individually to each reaction
F Primer .4 μL 2.4 μL
R Primer .4 μL 2.4 μL
Polymerase .2 μL 1.2 μL
H2O 15.6 μL 93.6 μL

Primers:10 micromolar

VF2, VR, ArsR F RFC10, ArsR R RFC10 (ArsR for positive control, separate reaction without verification primers, e. coli genome as template)

Samples:

  1. J23100B0034ArsRSDM2B0015pSB1C3 1
  2. J23100B0034ArsRSDM2B0015pSB1C3 2
  3. J23100B0034ArsRSDM2B0015pSB1C3 3
  4. + control (ArsR)
  5. - control (H2O)

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

12:45 PM

ArsB PCR

Ingredient Amount Mastermix (6 reactions)
10X Buffer 2 μL 12 μL
10 mM dNTPs .4 μL 2.4 μL
Template 1 μL (20ng) 6 μL
F Primer .4 μL added individually to each reaction
R Primer .4 μL added individually to each reaction
Polymerase .2 μL 1.2 μL
H2O 15.6 μL 93.6 μL

Samples:

  1. ArsB 1
  2. ArsB 2
  3. ArsB 3
  4. + control (ArsR)
  5. - control (H2O)

Primers: 10 micromolar

ArsBF3.0 ArsBR3.0 ArsRRFC10 ArsRFRFC10

Program:

95°C 3 min
95°C 30 sec
53°C 30 sec
72°C 90 sec
go to step 2 30X
72°C 3 min
hold at 4°C

3:25 PM

Gel

Lane Sample Amount
1 1 Kb Ladder 1 μL
2 J23100B0034ArsRSDM2B0015pSB1C3 1 1 μL dye + 5 μL sample
3 J23100B0034ArsRSDM2B0015pSB1C3 2 1 μL dye + 5 μL sample
4 J23100B0034ArsRSDM2B0015pSB1C3 3 1 μL dye + 5 μL sample
5 Negative 1 μL dye + 5 μL sample
6 ArsB 1 1 μL dye + 5 μL sample
7 ArsB 2 1 μL dye + 5 μL sample
8 ArsB 3 1 μL dye + 5 μL sample
9 + control 1 μL dye + 5 μL sample
10 - control 1 μL dye + 5 μL sample

UIUC 9.23.10 j23arsr in cap.jpg

4:00 PM

glycerol stocks

900 μL culture, 900 μL 50% glycerol for J23100B0034ArsRSDM2B0015pSB1C3 1 and J23100B0034ArsRSDM2B0015pSB1C3 2.