IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/08/02

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8/2/10

10:00 AM

got a good amount of colonies, a few white colonies on all plates. will pick for 5 mL overnights this afternoon.

5 mL overnights of ArsR-B0015-pSB1A2 spilled, 1 tube also appeared green...

made glycerol stock of ArsR-B0015-pSB1A2 colonies 1 and 2, meagan did miniprep

ArsR-B0015-pSB1A2 colony 1: ISB214

ArsR-B0015-pSB1A2 colony 2: ISB215

PCR Insert Verification

Run by Meagan

Ingredient Amount Mastermix (5 reactions)
10X buffer 2 μL 10 μL
10 mM dNTPs .4 μL 2 μL
Template 1 μL (20 ng) 5 μL
F Primer .2 μL 1 μL
R Primer .2 μL 1 μL
DMSO 1 μL 5 μL
Polymerase .3 μL 1.5 μL
H2O 14.9 μL 74.5 μL

Program:

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

Primers:

20 micromolar VR VF2

Samples:

LamBF2R

LamBF2R Green

ArsR-B0015-pSB1A2

negative control (no template)

10:25 PM

PCR

Ingredient Amount Mastermix (9 reactions)
10X Buffer 2 μL 18 μL
10 mM dNTPs .4 μL 3.6 μL
Template 1 μL (20 ng)
F primer .4 μL 3.6 μL
R Primer .4 μL 3.6 μL
DMSO 1 μL 9 μL
Polymerase .4 μL 3.6 μL
Water 13.4 μL 120.6 μL

Per reaction: 19 μL mastermix, 1 μL template

Program:

95°C 3 min
95°C 30 sec
48°C-68°C gradient 30 sec
72°C 2 min
go to step 2 30X
hold at 4°C

Primers:

ArsBF2

ArsBR2

Samples:

ArsB48°C

ArsB51°C

ArsB54°C

ArsB57°C

ArsB60°C

ArsB63°C

ArsB66°C

Negative control (no template)

1:05 PM

Gel

Top

Lane Sample Amount
1 1 Kb Ladder 1 μL
2 ArsB48 5 μL sample + 1 μL dye
3 ArsB51 5 μL sample + 1 μL dye
4 ArsB54 5 μL sample + 1 μL dye
5 ArsB57 5 μL sample + 1 μL dye
6 ArsB60 5 μL sample + 1 μL dye
7 ArsB63 5 μL sample + 1 μL dye
8 ArsB66 5 μL sample + 1 μL dye
9 Negative control 5 μL sample + 1 μL dye

Bottom

Lane Sample Amount
1 1 Kb Ladder 1 μL
2 LamBF2R 5 μL sample + 1 μL dye
3 LamBF2R green 5 μL sample + 1 μL dye
4 ArsR-B0015-pSB1A2 5 μL sample + 1 μL dye
5 negative control 5 μL sample + 1 μL dye
6 ArsRSDM1 (for band travel comparison) 5 μL sample + 1 μL dye

Image:UIUC_8.2.10ArsB_gradient,_verification_of_LamB_and_ArsR-terminator_labelled.jpg‎


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