IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/08/17

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8/17/2010

12:30PM
Gel of PCR from 8/16
1% Agarose gel run at 150V for ~25min. Lane
1. 1kb ladder
2. LamB EcoRI (5uL + 1uL dye)
3. LamB EcoRI (5uL + 1uL dye)
4. (-) Control (no template)

1:00PM
ArsR Re-Streak
2nd streak of ISB207 ArsR SDM2 pSB1A2

1:30PM
PCR Purification
PCR Purification of LamB EcoRI using Qiagen Kit

2:00PM
Digestion
Digesting LamB and pSB1C3 + RFP with EcoRI and SpeI

LamB PCR Product pSB1C3 + RFP
DNA 10uL 1 μL
H2O 32.5uL 41.5uL
Buffer 4 (NEB) 5uL 5uL
BSA 0.5uL 0.5uL
EcoRI 1uL 1uL
SpeI 1uL 1uL

37C Incubation for 15min.
80C Inactivation for 20min.

3:30PM
Ligation
Ligating pSB1C3 backbone with LamB using EcoRI and SpeI ends

pSB1C3 + LamB
H2O13uL
pSB1C3 (cut with EcoRI, SpeI)2uL
LamB (cut with EcoRI, SpeI)2uL
T4 DNA Ligase Buffer2uL
T4 DNA Ligase1uL

Room Temperature incubation for 10min.
42C for 20min -- Oops.
80C inactivation for 20min.

4:30
Transformation - Electroporation
Thaw cells --> +50uL cells + 4uL Ligation mixture --> Electroporate (The machine said Arc) --> +1mL SOC medium --> 37C recovery for 1hour.

Samples:
Control pUC19 Amp resistant plasmid
LamB pSB1C3 ligation

Plating
300uL on pre-warmed Cap and Amp plate.


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