IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/07/21

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7/21/10

Sold iGEM Dippin' Dots most of the day!!!


3:50 PM

Miniprepped yesterday's cultures of gas vesicles and promoters J23100/J23119. Diluted to 20 ng/μL for PCR.

5:00 PM

PCR

Ingredient Amount
10X buffer 2 μL
10 mM dTNPs .4 μL
Template 1 ul (20 ng)
V2R Primer .4 μL
VF Primer .4 μL
Polymerase .2 μL
H2O 15.6 μL

Samples:

J23100-Gas Vesicles-Kan Backbone

J23119-Gas Vesicles-Kan Backbone

Key1Tet (Steve)

Program:

95°C 2 min
95°C 30 sec
55 °C 30 sec
72 °C 6 min
Go to step 2 31X
72 °C 10 min
hold at 4°C

6:20 PM

Digestions

pRpoS438

DNA 16.39 μL
H2O 26.11 μL
Buffer 2 5 μL
BSA .5 μL
XbaI 1 μL
SpeI 1 μL

pArsRF1R1

DNA 17 μL
H2O 25.5 μL
Buffer 2 5 μL
BSA .5 μL
XbaI 1 μL
SpeI 1 μL

pSB1A2 (2X)

DNA 1.82 μL
H2O 40.68 μL
Buffer 2 5 μL
BSA .5 μL
XbaI 1 μL
SpeI 1 μL

Incubate at 37°C for 15 min, inactivate at 80°C for 20 min.

7:35 PM

Gel for Band Extraction

Lane Sample Amount
1 1 Kb Ladder 1 μL
2 pSB1A2 (1) 25 μL + 1 μL dye
3 pSB1A2 (1) 25 μL + 1 μL dye
4 pSB1A2 (2) 25 μL + 1 μL dye
5 pSB1A2 25 μL + 1 μL dye

Made 5 mL cultures of plates from this morning (ArsR-Amp): the NEB plate had much more growth compared to the plate we used our competent cells on.

UIUC plate ArsR.jpg

8:00 PM

Plated out Gas Vesicles-Promoters again on new Kan plates (200 μL).

9:05 PM

Band Extraction Concentrations (pSB1A2): 16.3 ng/μL and 12.6 ng/μL

9:15 PM

SAP

1.2 μL SAP buffer

.5 μL SAP

~10-12 μL pSB1A2 digest

incubated 20 min at 37°C, inactivated 15 min at 65°C

9:35 PM

Gel

Lane Sample Amount
1 1 kB 1 μL
2 J23100-gv-kan 5 μL + 1 μL dye
3 J23119-gv-kan 5 μL + 1 μL dye
4 Key1tet 5 μL + 1 μL dye

10:10 PM

pRpoS438-pSB1A2

H2O 13 μL
pRpoS 2 μL
pSB1A2 2 μL
Ligase Buffer 2 μL
Ligase 2 μL

pArsR-pSB1A2

H2O 13 μL
pArsR 2 μL
pSB1A2 2 μL
Ligase Buffer 2 μL
Ligase 2 μL

Room temp incubation 10 min, inactivation 80°C 20 min.

10:50 PM

Transformation

50 μL NEB electrocompetent 5α cells, 3 μL ligation product. electroporate, 1 mL LB, recover at 37°C for 1 hour.

11:50 PM

plated 200 ul cells on Amp plates.