IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/07/08

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7/8/10
11:00 AM

Colony Boil

25 ul H2O, swiped across plate to pick up a bunch of colonies. incubate at 95°C for 2 min. nanodropped, 444.6 ng/ul.

PCR Per Reaction: 32 ul Mastermix, 4 ul F Primer, 4 ul R Primer

Samples:

pArsR F1R1(540 bp)

pArsR F1R2 (547 bp)

pArsR F2R1 (293 bp)

pArsR F2R2 (300 bp)

ArsR 1

ArsR 2

negative control (no primers)

Primers (10mM):

pArsR F1

pArsR F2

pArsR R1

pArsR R2

ArsR F RFC10 EcoRI

ArsR R RFC10

Program:

2:05 PM

gel 90V

Blank gel again

2:30 PM

PCR #a million of LamB and ArsR and pArsR - NEW MIXTURE Per Reaction: 32 ul Mastermix, 4 ul F Primer, 4 ul R Primer

Samples: 1. LamB F1R 2. LamB F2R 3. ArsR 4. pArsR F1R1(540 bp) 5. pArsR F1R2 (547 bp) 6. pArsR F2R1 (293 bp) 7. pArsR F2R2 (300 bp) 8. negative control (no primers)

Program:


 * The gel for this PCR was run on Monday July 12th

3:25

digestions

pArsR

pSB1T3 Incubate at 37°C for 15 min, inactivate at 80°C for 20 min

PCR purified pSB1T3 and SAP: 2 ul SAP buffer, 1 ul SAP 37°C 10 min, 65°C 15 min.

4:45

PCR

Per Reaction: 32 ul mastermix, 4 ul F primer, 4 ul R primer

Primers:

pArsRF1

pArsRF2

pArsRR1

pArsRR2

ArsR F RFC10 EcoRI

ArsR R RFC10

Samples:

pArsR F1R1 540bp

pArsR F1R2 547bp

pArsR F2R1 293 bp

pArsR F2R2 300 bp

ArsR

Negative control (no primers

Program

SAP Backbone

2.6 ul SAP buffer

1.3 ul SAP

Incubate at 37°C for 10 min, inactivate at 65°C for 15 min.

Gel

8:40 PM

Band Extraction Zymogen Kit

pSB1T3 (7/1) and pArsR (7/7). the other samples didn't show up on gel.

Concentrations post-band extraction:

pSB1T3 8 ng/ul

pArsR 20 ng/ul

9:20 PM

Ligations Incubate at 14°C overnight, inactivate at 80°C for 20 min tomorrow.


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