IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/07/27

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7/27/10

9:50 AM

PCR cleanup:

LamBF1R: 5 ng/μL LamBF2R: 10 ng/μL

10:30 AM

Digestions:

LamBF1R

DNA 20 μL (116 ng)
H2O 22.5 μL
Buffer 4 5 μL
BSA .5 μL
XbaI 1 μL
SpeI 1 μL

lamBF2R

DNA 20 μL (204 ng)
H2O 22.5 μL
Buffer 4 5 μL
BSA .5 μL
XbaI 1 μL
SpeI 1 μL

pSB1C3

DNA 5. μL (225 ng)
H2O 42 μL
Buffer 4 5 μL
BSA .5 μL
XbaI 1 μL
SpeI 1 μL

incubate at 37°C for 15 min, inactivate at 80°C for 20 min.

12:00 PM

Gel

1%, TAE, 175V

lane Sample Amount
1 1 Kb ladder 1 μL
2 LamBF1R (oops..) 25 μL sample + 5 μL dye
3 pSB1C3 digest 1 25 μL sample + 5 μL dye
4 pSB1C3 digest 1 25 μL sample + 5 μL dye
5 pSB1C3 digest 2 25 μL sample + 5 μL dye
6 pSB1C3 digest 2 25 μL sample + 5 μL dye

Image:UIUC_7.27.10_backbones_digest_and_lamB_digest_(accident...).jpg

12:45 PM

Band Extraction:

pSB1C3 digest 1: 1.2 ng/μL

pSB1C3 digest 2: 1.9 ng/μL

1:20 PM

SAP

1.2 μL SAP buffer

.5 μL SAP

Incubate at 37°C for 20 min, inactivate at 65°C for 15 min.

2:25 PM

Ligations

LamBF1R-pSB1C3

H20 13 μL
LamBF1R 2 μL
pSB1C3 2 μL
10X Ligase Buffer 2 μL
Ligase 1 μL

LamBF2R-pSB1C3

H20 13 μL
LamBF2R 2 μL
pSB1C3 2 μL
10X Ligase Buffer 2 μL
Ligase 1 μL

Incubate at room temperature for 10 min, inactivate at 80°C for 20 min.

3:10 PM

Transformation and Plating

100 μL cells, 3 μL ligation product. electroporate. Add 1 mL SOC, recover at 37°C for 1 hour.

Plated 200 μL on CAP plates at 4:15 PM


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