IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/08/03

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8/3/10

9:45 AM

Digestions

ArsR-B0015

DNA 2 μL
H2O 39.5 μL
Buffer 4 5 μL
BSA .5 μL
XbaI 1 μL
PstI 2 μL


B0034

DNA .54 μL
H2O 41.96 μL
Buffer 4 5 μL
BSA .5 μL
EcoRI 1 μL
SpeI 1 μL


pSB1C3

DNA 1.11 μL
H2O 40.4 μL
Buffer 4 5 μL
BSA .5 μL
EcoRI 1 μL
PstI 2 μL

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

10:45 AM

Miniprep

Promega Kit

Concentrations:

pRpoS438 colony 1: 214.4 ng/μL

pRpoS438 colony 2: 380.3 ng/μL

pArsRF1R2 colony 1: 246.9 ng/μL

pArsRF1R2 colony 2: 456.2 ng/μL

11:10 PM

Ligations

ArsR-B0015 2 μL
B0034 2 μL
pSB1C3 2 μL
H2O 11 μL
Ligase Buffer 2 μL
Ligase 1 μL

Incubate at room temperature for 10 min, inactivate at 80°C for 20 min.

12:05 PM

Transformation/Plating 100 μL cells, 3 μL Ligation in cuvette. electroporate and add 1 mL SOC. Recover at 37°C for 1 hour.

Plated 100 μL on 1 half of a cap plate, 50 μL on the other half of the plate at 1:10 PM

12:40 PM

PCR

Verification of insert

Ingredient Amount Mastermix (6 reactions)
10X Buffer 2 μL 12 μL
10mM dNTPs .4 μL 2.4 μL
Template 1 μL (20 ng) added individually to each reaction
F Primer .2 μL 1.2 μL
R Primer .2 μL 1.2 μL
Polymerase .2 μL 1.2 μL
H2O 16 μL 96 μL

Per Reaction: 19 μL mastermix, 1 μL template.

Samples:

pRpoS438 colony 1

pRpoS438 colony 2

pArsRF1R2 colony 1

pArsRF1R2 colony 2

negative control (no template)

Primers:

20 micromolar

VF2

VR

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

3:05 PM

Gel

Lane Sample Amount
1 1 Kb Ladder 1 μL
2 pRpoS438 1 5 μL sample, 1 μL dye
3 pRpoS438 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 negative control 5 μL sample, 1 μL dye
7-10 Tom/Amanda's samples N/A

Image:UIUC_8.3.10_promoter_verification.jpg‎

started new 5 mL cultures of pArsRF1R2 and LamBF1R for verification PCR tomorrow. Will re-run today's samples with new pArsR and LamBF1R samples as well as old.



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