IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/07/05

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7/5/10
12:15 PM

PCR

add 36 ul mastermix and 4 ul primers to each reaction.

Program:

Samples:

1. PAL

2. ArsB

3. PAL negative control (no primers)

4. ArsB negative control (no primers

Program:

Samples:

1. ArsR

2. ArsR

Primers:

ArsR R RFC10

ArsR F RFC10 EcoRI

Gel Ran at 175V, got a blank gel.

8:00PM

Re-diluted primers to 10 micromolar, ran same PCR again. This time with two samples for ArsR as well. ran at 175 V



Digestion ArsR

pSB1A2 (6/30)

Incubate for 15 min at 37°C

Inactivate for 20 min at 80°C

9:45 PM

PCR Purification

Ligation NB: used new invitrogen ligase and buffer.

Incubate at room temp for 10 minutes, then inactivate at 80°C for 20 minutes.

10:30 PM

Streaked out some new e. Coli on LB for colony boils

11:00 PM

Transformation

100 ul cells and 5 ul ligation product, electroporated and added 1 mL LB. Recovered for 1 hour at 37°C. Plated 100 ul, 200 ul and 300 ul on separate Amp plates.


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