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From 2010.igem.org

August 3/2010

(In Lab: HB)

Objective: To restrict pBAD, sRBS, mRBS, TetR, dT and pTet for the assembly of pBAD-sRBS-TetR-dT-pTet

Method: Used Restriction of Plasmid DNA protocol.

• A front verctor was made made in sRBS ,mRBS, dT plasmids using EcoRI and Xbal enzymes
• pDNA that was cut out of plasmid for a front vector was pBAD, TetR, dT and pLacI using EcoRI and SpeI enzymes
• A back vector was made in sRBS and mRBS plasmids with PstI and SpeI
• pDNA that was cut out of plasmid for a back vector was TeTR and it was restricted with XbaI and PstI
  

Construct	pDNA	buffer	Enzymes
pBAD-sRBS/mRBS	pBAD	Red	EcoRI and SpeI
pBAD-sRBS/mRBS	sRBS	Orange	XbaI and EcoRI
pBAD-sRBS/mRBS	mRBS	Orange	XbaI and EcoRII
sRBS/mRBS-TetR	sRBS	Red	PstI and SpeI
sRBS/mRBS-TetR	mRBS	Red	PstI and SpeI
sRBS/mRBS-TetR	TetR	Tango	XbaI and PstI
TetR-dT	TetR	Red	EcoRI and SpeI
TetR-dT	dT	Orange	XbaI and EcoRI
dT-pTet	dT	Red	EcoRI and SpeI
dT-pTet	pTet	Orange	XbaI and EcoRI
pLAcI-sRBS/mRBS	pLacI	Red	EcoRI and SpeI
pLAcI-sRBS/mRBS	sRBS	Orange	XbaI and EcoRI
pLAcI-sRBS/mRBS	mRBS	Orange	XbaI and EcoRI
Mms6-PET28(a)	PET28(a)	Orange	NotI

• For all reactions
  • 158 (µL) MilliQ H2O
  • 10 (µL) Buffer
  • 0.5(µL) of each enzyme
  • 10 (µL) pDNA

Restriction was incubated for 1 hour at 37 0C