Wisconsin-Madison/28 August 2009
From 2009.igem.org
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August 28 2009
cloning with two single digests:
ProU PCR product with RBS/RFP
ProU PCR product with PSB1A2
ProU PCR product with GsSDMT
NudF with PSB1A2
Sequential Digestion
First Digestion
50uL / DNA / Buffer / H2O / Enzyme
ProU / 40 / 5 / 4 / EcoR I 1
nudF / 40 / 5 / 4 / XbaI 1.5
metK / 40 / 5 / 4 / XbaI 1.5
Hawaii / 40 / 5 / 4 / XbaI 1.5
RFP / 40 / 5 / 4 / EcoR I 1
Gssdmt / 40 / 5 / 4 / EcoR I 1
pSB1A2 / 40 / 5 / 4 / XbaI 1.5
PCR clean-up
Second Digestion
50uL / DNA / Buffer / H2O / Enzyme
ProU / 40 / 5 / 4 / Spe I 1
nudF / 40 / 5 / 4 / Pst 1.5
metK / 40 / 5 / 4 / Pst 1.5
Hawaii / 40 / 5 / 4 / Pst 1.5
RFP / 40 / 5 / 4 / XabI 1
Gssdmt / 40 / 5 / 4 / XbaI 1
pSB1A2 / 40 / 5 / 4 / PstI 1.5
Ran gel
Ligation