IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/08/11

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8/11/10

11:00 AM

Digestion

LamBF1R

DNA 28 μL (~112 ng)
H20 14.5 μL
Buffer4 5 μL
BSA .5 μL
SpeI 1 μL
XbaI 1 μL

LamBF2R

DNA 28 μL (~112 ng)
H20 14.5 μL
Buffer4 5 μL
BSA .5 μL
SpeI 1 μL
XbaI 1 μL

pSB1C3

DNA .5 μL (225 ng)
H20 42 μL
Buffer4 5 μL
BSA .5 μL
SpeI 1 μL
XbaI 1 μL

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

12:30 PM

Gel

100V, low melting temp agarose. 1%, TAE.

Lane Sample Amount
1 1 Kb ladder 1 μL
2/3/4 pSB1C3 digest 50 μL sample, 10 μL dye

1:25 PM

Gel Extraction

Zymogen kit. pSB1C3 from gel.

1:55 PM

SAP

pSB1C3: 2 μL SAP 10 Buffer: 2.28 μL SAP: .5 μL

incubate for 20 min at 37°C, inactivate for 15 min at 65°C

2:35 PM

Ligation

pSB1C3-LamBF1R

LH2O 13 μL
pSB1C3 2 μL
LamBF1R 2 μL
Ligase Buffer 2 μL
Ligase 1 μL

pSB1C3-LamBF2R

LH2O 13 μL
pSB1C3 2 μL
LamBF2R 2 μL
Ligase Buffer 2 μL
Ligase 1 μL

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

3:25 PM

Transformation

thaw cells. add 50 μL cells and 4 μL ligation to a tube. put the tube on ice for 10 min, then shock at 42°C for 45 seconds. put the sample back on ice for 2 minutes. recover in 1 mL SOC for 1 hour at 37°C shaking.

Transformed pSB1C3-LamBF1R, pSB1C2 LamBF2R, pArsRF1R2 2' SDM, pArsRF1R2 1 SDM, pArsRF1R2 2 SDM, and pRpoS438 2 SDM.

plated 300 μL on pre-warmed CAP plates.



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