IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/07/29

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7/29/10

10:30 AM

prepared ArsR SDM1 and ArsR SDM2 samples for sequencing:

3 μL VF2 stock primer 3 μL VR stock primer 4.5 μL Template (200 ng/μL) 19.5 μL H2O

11:00

Digestions

ArsR SDM1

DNA 2.4 μL
H2O 40.1 μL
Buffer4 5 μL
BSA .5 μL
EcoRI 1 μL
SpeI 1 μL

B0015

DNA 1.6 μL
H2O 40.9 μL
Buffer4 5 μL
BSA .5 μL
XbaI 1 μL
PstI 1 μL

pSB1A2

DNA 1.8 μL
H2O 39.7 μL
Buffer4 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.

Ligations

H2O 11 μL
ArsR SDM 1 2 μL
B0015 2 μL
pSB1A2 2 μL
Ligase Buffer 2 μL
Ligase 1 μL

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

12:30 PM

Transformation

100 μL cells, 3 μL Ligation, electroporate, add 1 mL SOC, recover for 1 hour at 37°C

Plated 200 μL at 1:45 PM on Amp plates.

1:15 PM

PCR

Ingredient Amount Mastermix (4 reactions)
10X buffer 2 μL 8 μL
10mM dNTPs .4 μL 1.6 μL
Template 1 μL (20 ng) 4 μL
F Primer .4 μL added individually to each reaction
R Primer .4 μL added individually to each reaction
Polymerase .2 μL .8 μL
H2O 15.6 μL 62.4 μL

Per reaction: 19.2 μL mastermix, .4 μL F Primer, .4 μL R Primer.

Primers:

pArsR RFC10F-502 EcoRI

pArsR R1

pArsR R2

pRpoS RFC0F-438 EcoRI

pRpoS R

Samples:

pArsR F1R1

pArsR F1R2

pRpoS F-438R

Program:

95°C 3 min
95°C 30 sec
53°C 30 sec
72°C 90 sec
go to step 2 31X
72°C 5 min
hold at 4°C

4:00 PM

Gel

lane sample amount
1 1 Kb ladder 1 μL
2 pArsRF1R1 (540bp) 5 μL + 1 μL dye
3 pArsRF1R2 (547bp) 5 μL + 1 μL dye
4 pRpoS (438 bp) 5 μL + 1 μL dye
5 Tom 1 5 μL + 1 μL dye
6 Tom 2 5 μL + 1 μL dye
7 negative control 5 μL + 1 μL dye

UIUC 7.29.10 promoters.jpg

Made 5 mL culture of LamB colonies from yesterday. incubated at 37°C for miniprep tomorrow.