IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/07/21

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7/21/10
Sold iGEM Dippin' Dots most of the day!!!

3:50 PM

Miniprepped yesterday's cultures of gas vesicles and promoters J23100/J23119. Diluted to 20 ng/μL for PCR.

5:00 PM

PCR

Samples:

J23100-Gas Vesicles-Kan Backbone

J23119-Gas Vesicles-Kan Backbone

Key1Tet (Steve)

Program:

6:20 PM

Digestions

pRpoS438

pArsRF1R1

pSB1A2 (2X) Incubate at 37°C for 15 min, inactivate at 80°C for 20 min.

7:35 PM

Gel for Band Extraction

Made 5 mL cultures of plates from this morning (ArsR-Amp): the NEB plate had much more growth compared to the plate we used our competent cells on.



8:00 PM

Plated out Gas Vesicles-Promoters again on new Kan plates (200 μL).

9:05 PM

Band Extraction Concentrations (pSB1A2): 16.3 ng/μL and 12.6 ng/μL

9:15 PM

SAP

1.2 μL SAP buffer

.5 μL SAP

~10-12 μL pSB1A2 digest

incubated 20 min at 37°C, inactivated 15 min at 65°C

9:35 PM

Gel

10:10 PM

pRpoS438-pSB1A2

pArsR-pSB1A2 Room temp incubation 10 min, inactivation 80°C 20 min.

10:50 PM

Transformation

50 μL NEB electrocompetent 5α cells, 3 μL ligation product. electroporate, 1 mL LB, recover at 37°C for 1 hour.

11:50 PM

plated 200 ul cells on Amp plates.


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