IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/08/09

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8/9/10

10:00A AM

PCR verification AGAIN!!

Ingredient Amount Mastermix (15 reactions)
10X Buffer 2 μL 30 μL
10 mM dNTPs .4 μL 6 μL
Template 1 μL (20 ng) added individually to each reaction
F Primer .4 μL 6 μL
R Primer .4 μL 6 μL
DMSO 1 μL 15 μL
Polymerase .3 μL 4.5 μL
H2O 14.5 μL 217.5 μL

Per reaction: 19 μL mastermix, 1 μL template

Progam:

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

Samples:

  1. B0034ArsRSDM1B0015pSB1C3 1
  2. B0034ArsRSDM1B0015pSB1C3 2
  3. B0034ArsRSDM1B0015pSB1C3 3 (1')
  4. B0034ArsRSDM1B0015pSB1C3 4 (2')
  5. LamBF1R 1'
  6. LamBF1R 2'
  7. LamBF2R
  8. LamBF2R Green
  9. pArsR F1R2 1'
  10. pArsRF1R2 2'
  11. pArsRF1R2 1
  12. pArsRF1R2 2
  13. Negative control (no primer) template B0034ArsRSDM1B0015pSB1C3 1
  14. Negative control (no template)

Primers: VF2, VR 10 micromolar

1:45 PM

Gel

Top

Lane Sample Amount
1 1 Kb Ladder 1 μL
2 B0034ArsRSDM1B0015pSB1C3 1 5 μL sample, 1 μL dye
3 B0034ArsRSDM1B0015pSB1C3 2 5 μL sample, 1 μL dye
4 B0034ArsRSDM1B0015pSB1C3 3 (1') 5 μL sample, 1 μL dye
5 B0034ArsRSDM1B0015pSB1C3 4 (2') 5 μL sample, 1 μL dye
6 LamBF1R 1' 5 μL sample, 1 μL dye
7 LamBF1R 2' 5 μL sample, 1 μL dye
8 LamBF2R 5 μL sample, 1 μL dye
9 LamBF2R Green 5 μL sample, 1 μL dye
10 pArsRF1R2 1' 5 μL sample, 1 μL dye

Bottom:

Lane Sample Amount
1 1 Kb Ladder 1 μL
2 pArsRF1R2 2' 5 μL sample, 1 μL dye
3 pArsRF1R2 1 5 μL sample, 1 μL dye
4 pArsRF1R2 2 5 μL sample, 1 μL dye
5 Negative control, no primer 5 μL sample, 1 μL dye
6 negative control, no template 5 μL sample, 1 μL dye

Image: UIUC_8.9.10_verification.jpg

2:45 PM

made sequencing reactions:

1.5 μL ArsRSDM2

2 μL 10 micromolar VF2

6.5 μL H2O

Made 2 reactions in case sample needed to be re-run.

3:35 PM

Recieved and suspended new primers to 100 micromolar. Then diluted aliquots to 10 micromolar.


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