IGEM:University of Illinois Urbana Champaign/2009/Notebook/Bioware 2010 Arsenic Bioremediation/2010/07/09

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7/9/10

Colony Boil

50 ul H2O, 3 colonies. gave 25 ng/ul DNA.

PCR

Ingredient Amount Mastermix (9 reactions)
10X Buffer 2 ul 18 ul
10 mM dNTPs .4 ul 3.6 ul
F primer .4 ul added individually to each reaction
R primer .4 ul added individually to each reaction
Template 4 ul (100ng) 36 ul
Polymerase .2 ul 1.8 ul
H2O 12.6 ul 113.4 ul

Per Reaction: 19.2 ul mastermix, .4 ul F Primer, .4 ul R primer

Primers

pArsR F1

pArsR F2

pArsR R1

pArsR R2

ArsR F RFC10 EcoRI

ArsR R RFC10

ArsR R fusion BSAI

ArsR F Fusion BSAI

LamB F1 RFC10 XbaI

LamB F2 RFC10 XbaI

LamB R RFC10 SpeI

Samples

LamBF1R

LamBF2R

ArsR Fusion

ArsR

pArsR F1R1

pArsR F1R2

pArsR F2R1

pArsR F2R2

Negative control (no primers)

Program

95°C 3 min
95°C 30 sec
53°C 30 sec
72°C 90 sec
go to step 2 31X
72°C 5 min
hold at 4°C

Meagan did transformation of yesterday's ligation at 3:55 PM, incubated for an hour at 37°C and plated 200 ul on Tet plate at 4:55 PM.