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Team:Lethbridge/Notebook/Lab Work/August
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(New page: ==August 3/2010== (In Lab: HB)<br> <b>Objective:</b> To restrict pBAD, sRBS, mRBS, TetR, dT and pTet for the assembly of pBAD-sRBS-TetR-dT-pTet<br> <b>Method:</b> Used [[Team:Lethbridge/...) |
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* A back vector was made in sRBS and mRBS plasmids with PstI and SpeI | * 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 <br> | * pDNA that was cut out of plasmid for a back vector was TeTR and it was restricted with XbaI and PstI <br> | ||
| + | |||
| + | <table><table border ="3"> | ||
| + | <tr><td><b>Construct</b></td><td><b>pDNA</b></td><td><b>buffer</b></td><td><b>Enzymes</b></td></tr> | ||
| + | <tr><td>pBAD-sRBS/mRBS</td><td>pBAD</td><td>Red</td><td>EcoRI and SpeI</td></tr> | ||
| + | <tr><td>pBAD-sRBS/mRBS</td><td>sRBS</td><td>Orange</td><td>XbaI and EcoRI</td></tr> | ||
| + | <tr><td>pBAD-sRBS/mRBS</td><td>mRBS</td><td>Orange</td><td>XbaI and EcoRII</td></tr> | ||
| + | <tr><td>sRBS/mRBS-TetR</td><td>sRBS</td><td>Red</td><td>PstI and SpeI</td></tr> | ||
| + | <tr><td>sRBS/mRBS-TetR</td><td>mRBS</td><td>Red<td>PstI and SpeI</td></tr> | ||
| + | <tr><td>sRBS/mRBS-TetR</td><td>TetR</td><td>Tango</td><td>XbaI and PstI</td></tr> | ||
| + | <tr><td>TetR-dT</td><td>TetR</td><td>Red</td><td>EcoRI and SpeI</td></tr> | ||
| + | <tr><td>TetR-dT</td><td>dT</td><td>Orange</td><td>XbaI and EcoRI</td></tr> | ||
| + | <tr><td>dT-pTet</td><td>dT</td><td>Red</td><td>EcoRI and SpeI</td></tr> | ||
| + | <tr><td>dT-pTet</td><td>pTet</td><td>Orange</td><td>XbaI and EcoRI</td></tr> | ||
| + | <tr><td>pLAcI-sRBS/mRBS</td><td>pLacI</td><td>Red</td><td>EcoRI and SpeI</td></tr> | ||
| + | <tr><td>pLAcI-sRBS/mRBS</td><td>sRBS</td><td>Orange</td><td>XbaI and EcoRI</td></tr> | ||
| + | <tr><td>pLAcI-sRBS/mRBS</td><td>mRBS</td><td>Orange</td><td>XbaI and EcoRI</td></tr> | ||
| + | <tr><td>Mms6-PET28(a)</td><td>PET28(a)</td><td>Orange</td><td>NotI</td></tr> | ||
| + | </table><br> | ||
| + | * 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 | ||
Revision as of 01:31, 25 August 2010
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
