IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/07/19

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7/19/10

10:00 AM

Francis finished up genomic prep for me using the DNeasy kit. got a concentration of 98 ng/μL

diluted to 100 μL aliquots at 20ng/μL

3:20 PM

PCR

Ingredient Amount Mastermix (14 reactions)
10X buffer 2 μL 28 μL
10 mM dNTPs .4 μL 5.6 μL
Template 20 ng (1 μL) 14 μL
F Primer .4 μL 5.6 μL
R Primer .4 μL 5.6 μL
Polymerase .2 μL 2.8 μL
H2O 15.6 μL 218.4 μL

Per Reaction: 19.2 μL mastermix, .4 μL F primer, .4 μL reverse primer

samples

pRpoS438

pRpoS232

ArsB

LamB F1R

LamB F2R

ArsR fusion

ArsR

pArsR F1R1

pArsR F1R2

pArsR F2R1

pArsR F2R2

Negative Control (no primers)

Primers

pRpoSF438

pRpoSF232

pRpoSR

ArsBF2

ArsBR2

ArsR F RFC10 EcoRI

ArsR R RFC10

ArsR R fusion BSAI

ArsR F fusion BSAI

pARsR F1

pArsR F2

pArsR R1

pArsR R2

LamB F1

LamB F2

LamB R

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

6:30 PM

Gel

Top:

Lane Sample Amount
1 forgot to load 1 kB ladder N/A
2 pRpoS438 5 μL sample, 1 μL dye
3 pRpoS 5 μL sample, 1 μL dye
4 ArsB 5 μL sample, 1 μL dye
5 LamB F1R 5 μL sample, 1 μL dye
6 LamB F2R 5 μL sample, 1 μL dye
7 ArsR Fusion 5 μL sample, 1 μL dye
8 ArsR 5 μL sample, 1 μL dye
9 pArsRF1R1 5 μL sample, 1 μL dye
10 pArsRF2R2 5 μL sample, 1 μL dye
Lane Sample Amount
1 forgot to load 1 Kb ladder N/A
2 pArsR F2R1 5 μL sample, 1 μL dye
3 pArsR F2R2 5 μL sample, 1 μL dye
4 negative control 5 μL sample, 1 μL dye
5 OmpA (Francis) 5 μL sample, 1 μL dye
6 Francis' Sample 5 μL sample, 1 μL dye
7 Francis' Sample 5 μL sample, 1 μL dye

UIUC 7.19.10 everything from genomic prep.jpg

7:00 PM

Digestions

J23100

DNA .5 μL
H2O 42 μL
Buffer 2 5 μL
BSA .5 μL
EcoRI 1 μL
SpeI 1 μL

J23119

DNA 2.64 μL
H2O 39.86 μL
Buffer 2 5 μL
BSA .5 μL
EcoRI 1 μL
SpeI 1 μL

gas vesicles

DNA 1.16 μL
H2O 41.34 μL
Buffer 2 5 μL
BSA .5 μL
XbaI 1 μL
PstI 1 μL

pSB1K3

DNA 1.56 μL
H2O 40.94 μL
Buffer 2 5 μL
BSA .5 μL
EcoRI 1 μL
PstI 1 μL

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

8:20

Ligations

J23100-gas vesicles-pSB1K3

H2O 11 μL
J23100 2 μL
Gas vesicles 2 μL
pSB1K3 2 μL
Ligase Buffer 2 μL
Ligase 1 μL


J23119-gas vesicles-pSB1K3

H2O 11 μL
J23119 2 μL
Gas vesicles 2 μL
pSB1K3 2 μL
Ligase Buffer 2 μL
Ligase 1 μL

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

9:15 PM

Transformations and Plating

100 μL cells, 3 μL ligation product, electroporate, add 1 mL LB, recover at 37°C for 1 hour.

Plated 200 μL cells on Kan plates at 10:15 PM.