IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/08/05

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8/5/10

9:45 AM

miniprepped B0034-ArsR-B0015-pSB1C3 overnights using the promega kit. The colony 2 overnight may have spilled or something since it looked like we were missing about 3 mLs.

concentrations:

B0034-ArsR-B0015-pSB1C3 colony 1: 767.6 ng/μL

B0034-ArsR-B0015-pSB1C3 colony 2: 376.3 ng/μL

Dilutions to 20 ng/μL:

B0034-ArsR-B0015-pSB1C3 colony 1 1.3 μL Sample 48.7 μL water
B0034-ArsR-B0015-pSB1C3 colony 2 2.6 μL Sample 47.4 μL H2O

also got good growth on steak plates of the 1/2 colonies from B0034-ArsR-B0015-pSB1C3.

11:00 AM

PCR

Ingredient Amount Mastermix (12 reactions)
10X buffer 2 μL 24 μL
10 mM dNTPs .4 μL 4.8 μL
Template 1 μL (20 ng) added individually to each reaction
F Primer .2 μL 2.4 μL
R Primer 2 μL 2.4 μL
Polymerase .3 μL 3.6 μL
DMSO 1 μL 12 μL
H2O 14.9 μL 178.8 μL

Per Reaction: 19 μL mastermix, 1 μL template

Primers: VF, V2R 20 micromolar

Program:

95°C 3 min
95°C 30 sec
54°C 30 sec
72°C 2 min
go to step 2 30X
72°C 3 min
hold at 4°C

Samples:

  1. B0034-ArsR-B0015-pSB1C3 colony 1
  2. B0034-ArsR-B0015-pSB1C3 colony 2
  3. LamBF1R 1'
  4. LamBF1R 2'
  5. pArsRF1R2 1'
  6. pArsRF1R2 2'
  7. LamBF2R
  8. LamBF2R green
  9. pArsRF1R2 1
  10. pArsRF1R2 2
  11. Negative control (no template)
  12. Negative control (no primers, template=pArsRF1R2)


3:30 PM

gel

Top:

Lane Sample Amount
1 1 Kb Ladder 1 μL
2 pArsRF1R2 1' 5 μL sample + 1 μL dye
3 pArsRF1R2 2' 5 μL sample + 1 μL dye
4 pArsRF1R2 1 5 μL sample + 1 μL dye
5 pArsRF1R2 2 5 μL sample + 1 μL dye
6 1 Kb ladder 1 μL


Bottom:

Lane Sample Amount
1 1 Kb Ladder 1 μL
2 GolT 1 (Tom) 5 μL sample + 1 μL dye
3 Gol T 2 (Tom) 5 μL sample + 1 μL dye
4 Gol T 3 (Tom) 5 μL sample + 1 μL dye
5 Gol T 4 (Tom) 5 μL sample + 1 μL dye
6 Gol S (Tom 5 μL sample + 1 μL dye


Image:UIUC_8.5.10_pArsR_verification_and_Tom's.jpg‎

Lane Sample Amount
1 1 Kb Ladder 1 μL
2 B0034-ArsR-B0015-pSB1C3 colony 1 5 μL sample + 1 μL dye
3 B0034-ArsR-B0015-pSB1C3 colony 2 5 μL sample + 1 μL dye
4 LamBF1R 1' 5 μL sample + 1 μL dye
5 LamBF1R 2' 5 μL sample + 1 μL dye
6 LamBF2R 5 μL sample + 1 μL dye
7 LamBF2R green 5 μL sample + 1 μL dye
8 ArsR SDM 2 5 μL sample + 1 μL dye
9 No Template 5 μL sample + 1 μL dye
10 No Primer 5 μL sample + 1 μL dye

Image:UIUC_8.5.10_verification.jpg‎

Made 5 mL cultures of B0034-ArsRSDM1-B0015-pSB1C3 colonies from streak plates since we saw multiple bands in the verification gel.

4:15 PM

PCR purified ArsRSDM2 to send for sequencing tomorrow (qiagen kit). concentration=20 ng/μL.


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