IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/08/19

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8/19/10

10:00 AM

PCR

Ingredient Amount Mastermix (4 reactions
10X Buffer 2 μL 8 μL
10 mM dNTPs .4 μL 1.6 μL
Template .9 μL (18 ng) 3.6 μL
F Primer .4 μL added individually to each reaction
R Primer .4 μL added individually to each reaction
Polymerase .3 μL 1.2 μL
H2O 14.6 μL 58.4 μL
DMSO 1 μL 4μL

Samples:

  • ArsB50
  • ArsB53
  • ArsB56
  • Negative Control (no primers)

Primers:

  • ArsBF2.0
  • ArsBR2.0

Program:

95°C 3 min
95°C 30 sec
50-56°C gradient 30 sec
72°C 2 min
go to step 2 32X
72°C 5 min
hold at 4°C

11:15 AM

Made glycerol stock of LamBF2

.9 ML 50% glycerol, .9 mL LamBF2 overnight.

11:20 AM

Digestion ArsRSDM2

DNA 2.7 μL (500ng)
H2O 39.8 μL
Buffer 4 5 μL
BSA .5 μL
EcoRI 1 μL
SpeI 1 μL

B0015

DNA 1.55 μL (500ng)
H2O 39.95 μL
Buffer 4 5 μL
BSA .5 μL
XbaI 1 μL
PstI 2 μL

pSB1T3

DNA 1 μL (500ng)
H2O 40.5 μL
Buffer 4 5 μL
BSA .5 μL
EcoRI 1 μL
PstI 2 μL

Incubate at 37 for 15°C min. Inactivate at 80°C for 20 min.

1:15 PM

Ligation

ArsRSDM2-B0015-pSB1T3

H2O 11 μL
pSB1T3 2 μL
ArsRSDM2 2 μL
B0015 2 μL
Ligase Buffer 2 μL
Ligase 1 μL

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

1:50 PM

Transformation

50 μL cells, 2 μL ligation product. 10 min on ice, 45 sec at 42°C, 2 min on ice. then add 1 mL SOC, recover for 1 hour at 37°C shaking.

2:20 PM

Gel

Lane Sample Amount
1 1 Kb Ladder 1 μL
2 ArsB50 5 μL sample, 1 μL dye
3 ArsB53 5 μL sample, 1 μL dye
4 ArsB56 5 μL sample, 1 μL dye

UIUC8.19.10ArsBlabeled.jpg

It's hard to see, but there are very faint bands around 1.3 Kb which is the expected size for ArsB

Made 5 mL Cultures (2 colonies from each plate section)of the following samples:

  • pArsRF1R2 1
  • pRpoS438 electroporated
  • pRpoS438 heat shock
  • ArsRSDM2-B0015-pSB1T3

ArsRSDM2-B0015-pSB1T3 plate had questionable looking colonies (hence re-doing yesterday's procedure today) but I picked what looked like colonies anyways.

No growth on pArsRF1R2 2 or pArsRF1R2 2' plates.


3:15 PM

Plating

Plated 300 μL transformation product on Tet plate

3:30 PM

PCR Cleanup

ArsB50: 4.8 ng/μL ArsB53: 6.1 ng/μL ArsB56: 6.3 ng/μL

Francis Miniprepped ArsRSDM2 and LamBF2 overnights, both at 250 ng/μL.