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 - RFPpSB1C3 - RFP pSB2K3 - E0033 pSB1T3 - RFP

11:10 AM

PCR 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

2:50 PM

Gel

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

pSB1T3

pSB1C3

pSB1C3

pArsRF1R1

ArsR

RpoS435

8:00 PM

Gel/Band Extraction

Zymogen Kit, 1% gel, TAE



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

pSB1T3 XbaI/SpeI-RpoS

pSB1T3 EcoRI/SpeI-ArsR Incubate 14°C Overnight. 80°C 20 min inactivation (7/15/10 8:45 AM)


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