Team:Lethbridge/Notebook/Lab Work/August

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Contents

August

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
ConstructpDNAbufferEnzymes
pBAD-sRBS/mRBSpBADRedEcoRI and SpeI
pBAD-sRBS/mRBSsRBSOrangeXbaI and EcoRI
pBAD-sRBS/mRBSmRBSOrangeXbaI and EcoRII
sRBS/mRBS-TetRsRBSRedPstI and SpeI
sRBS/mRBS-TetRmRBSRedPstI and SpeI
sRBS/mRBS-TetRTetRTangoXbaI and PstI
TetR-dTTetRRedEcoRI and SpeI
TetR-dTdTOrangeXbaI and EcoRI
dT-pTetdTRedEcoRI and SpeI
dT-pTetpTetOrangeXbaI and EcoRI
pLAcI-sRBS/mRBSpLacIRedEcoRI and SpeI
pLAcI-sRBS/mRBSsRBSOrangeXbaI and EcoRI
pLAcI-sRBS/mRBSmRBSOrangeXbaI and EcoRI
Mms6-PET28(a)PET28(a)OrangeNotI

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

Restriction was incubated for 1 hour at 370C


Harland to read over and make sure what i wrote is correct

August 3/2010 Evening

(In Lab:K.G J.S)

Objective: To run 1.5% agarose of restrictions: pBAD, sRBS, mRBS, TetR, dT and pTet for the assembly of pBAD-sRBS-TetR-dT-pTet

Method: Used a 1.5% agarose gel with 2 (µL) of loading dye and 10 (µL) of pDNA.

August 4/2010

(in Lab: JV)

Objective: PCR analysis of ligation product of aug 3/2010

will PCR: