IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/07/14

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7/14/10

11:00 AM

Miniprep for plasmid backbones:
pSB1A2 - RFP
pSB1C3 - RFP
pSB2K3 - E0033
pSB1T3 - RFP

11:10 AM

PCR

Ingredient Amount Mastermix (6 reactions)
10X Buffer 2 μL 12 μL
10mM dNTPs .4 μL 2.4 μL
Template 1 μL (20 ng) 6 μL
F Primer .4 μL added individually to each reaction
R Primer .4 μL added individually to each reaction
Polymerase .2 μL 1.2 μL
H2O 15.6 μL 93.6 μL

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

Samples

pRpoS438

pRpoS252

ArsB

Negative control (no primers)

Primers

pRpoSF438

pRpoSF252

pRpoSR1

ArsBF2

ArsBR2

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

2:50 PM

Gel

Lane Sample Amount
1 1 kb Ladder 1 μL
2 pArsR 438 5 μL Sample, 1 μL dye
3 pArsR 252 5 μL Sample, 1 μL dye
4 ArsR 5 μL Sample, 1 μL dye

UIUC 7.14.10 3pm PCR of RpoS and ArsB.jpg

6:15 PM

Nanodropped after PCR:

pArsRF1R2: 314.6 ng/μL

ArsR: 310.8 ng/μL

RpoS 438: 383.4 ng/μL

PCR purified samples (qiagen kit):

pArsRF1R2: 24.6 ng/μL

ArsR: 4.8 ng/μL

RpoS 438: 12.2 ng/μL

Digestions

pSB1T3

DNA 1 μL (500 nog
H2O 41.5 μL
Buffer 2 5 μL
BSA .5 μL
XbaI 1 μL
SpeI 1 μL

pSB1T3

DNA 1 μL (500ng)
H2O 41.5 μL
Buffer 2 5 μL
BSA .5 μL
XbaI 1 μL
EcoRI 1 μL

pSB1C3

DNA 1.1 μL (500 ng)
H2O 41.4 μL
Buffer 2 5 μL
BSA .5 μL
XbaI 1 μL
SpeI 1 μL

pSB1C3

DNA 1.1 μL (500 ng)
H2O 41.4 μL
Buffer 2 5 μL
BSA .5 μL
EcoRI 1 μL
SpeI 1 μL

pArsRF1R1

DNA 20 μL
H2O 22.5 μL
Buffer 2 5 μL
BSA .5 μL
XbaI 1 μL
SpeI 1 μL

ArsR

DNA 28 μL
H2O 14.5 μL
Buffer 2 5 μL
BSA .5 μL
EcoRI 1 μL
SpeI 1 μL

RpoS435

DNA 28 μL
H2O 14.5 μL
Buffer 2 5 μL
BSA .5 μL
XbaI 1 μL
SpeI 1 μL

8:00 PM

Gel/Band Extraction

Zymogen Kit, 1% gel, TAE

Lane Sample Amount
1 1 Kb Ladder 1 μL
2,3 pSB1T3 XbaI/SpeI each lane, 25 μL sample, 5 μL dye
4,5 pSB1T3 EcoRI/SpeI each lane, 25 μL sample, 5 μL dye
6,7 pSB1C3 XbaI/SpeI each lane, 25 μL sample, 5 μL dye
8,9 pSB1C3 each lane, 25 μL sample, 5 μL dye
10 1 Kb Ladder 1 μL

UIUC 7.14.10 9PM digest pSB1T3 pSB1C3.1 copy.jpg

Nanodrop after purification:

pSB1T3 XbaI/SpeI: 28.39 ng/μL

pSB1T3 EcoRI/SpeI: 20.3 ng/μL

pSB1C3 XbaI/SpeI: 18.2 ng/μL

pSB1C3 EcoRI/SpeI: 18.3 ng/μL

SAP SAP Buffer (10X) 1 ul SAP .5 ul H2O .5 ul

SAP-ed pSB1T3 XbaI/SpeI and pSB1C3 XbaI/SpeI. Incubate 20 min at 37°C, inactivate 15 min at 65°C. Flash Spin, remove DNA from top for ligations.

Ligation

pSB1T3 XbaI/SpeI-pArsR

H2O 13 μL
pSB1T3 XbaI/SpeI 2 μL
pArsR 2 μL
10X Ligase Buffer 2 μL
Ligase μL

pSB1T3 XbaI/SpeI-RpoS

H2O 13 μL
pSB1T3 XbaI/SpeI 2 μL
RpoS 2 μL
10X Ligase Buffer 2 μL
Ligase μL

pSB1T3 EcoRI/SpeI-ArsR

H2O 13 μL
pSB1T3 XbaI/SpeI 2 μL
ArsR 2 μL
10X Ligase Buffer 2 μL
Ligase μL

Incubate 14°C Overnight. 80°C 20 min inactivation (7/15/10 8:45 AM)