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Team:Lethbridge/Notebook/Lab Work/August
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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
| 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) 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
