IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/07/22

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7/22/10

Ordered new pRpoS438F and pArsRF1 primers so they have an EcoRI site instead o XbaI.

Lawns on gas vesicle/promoter plates again

No growth on pRpoS/pArsR plates

10:25 AM

miniprepped ArsR/pSB1A2 colonies.

concentrations:

ArsR-pSB1A2 colony 1: 288.2 ng/μL

ArsR-pSB1A2 colony 2: 323.9 ng/μL

Diluted each sample to 20 ng/μL for PCR

11:00 AM

PCR

Ingredient Amount Mastermix (4 reactions)
10X Buffer 2 μL 8 μL
10 mM dNTPs .4 μL 1.6 μL
Template 1 μL (20 ng) Added individually to each reaction
F Primer .2 μL .8 μL
R Primer .2 μL .8 μL
Polymerase .2 μL .8 μL
H2O 16 μL 64 μL

Per reaction:

18.8 μL mastermix, .2 μL F primer, .2 μL R primer, 1 μL template

Samples:

ArsR-pSB1A2 colony 1

ArsR-pSB1A2 colony 2

Negative control (no template)

Primers (20 micromolar)

VR

V2F

Program:

95°C 3 min
95°C 30 sec
55°C 30 sec
72°C 90 sec
go to step 2 31X
72°C 5 min
hold at 4°C

2:05 PM

Gel 1%, TAE 175V

Lane Sample Amount
1 1 Kb Ladder 1μL
2 ArsR-pSB1A2 colony 1 5 μL sample, 1 μL dye
3 ArsR-pSB1A2 colony 2 5 μL sample, 1 μL dye
4 negative control 5 μL sample, 1 μL dye

UIUC 7.22.10 arsR (1).jpg

3:15 PM

Site Directed Mutagenesis PCR

Modified from Quikchange kit (stratagene)

Ingredient Amount
10X Buffer 5 μL
10 mM dNTPs 1 μL
Template 1 μL (20ng)
Primer 1 .2 μL (~125 ng)
Primer 2 .2 μL (~125 ng)
Polymerase 1 μL
H2O 41.6 μL

Sample:

ArsR-pSB1A2 colony 1

Primers:

ArsR SDM 1 (100 micromolar)

ArsR SDM 2 (100 micromolar)

Program:

95°C 3 min
95°C 30 sec
66°C 1 min
72 1 min
go to step 2 12X
72 1 min
hold for 4°C