Team:Lethbridge/Notebook/Lab Work/August
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will PCR:<br> | will PCR:<br> | ||
+ | * <b>ligations</b> | ||
+ | ** pBAD-mRBS | ||
+ | ** pBAD-SRBS | ||
+ | ** SRBS-tetR | ||
+ | ** mRBS-TetR | ||
+ | ** dt-pTet | ||
+ | ** mms6-pET-28a | ||
+ | ** dt-pSBIC3 | ||
+ | ** pLacI-SRBS | ||
+ | |||
+ | * <b>controls</b> | ||
+ | ** pBAD | ||
+ | ** TetR | ||
+ | ** TetR | ||
+ | ** pLacI | ||
+ | ** Mms6 | ||
+ | |||
+ | <table border ="3"> | ||
+ | <tr><td> <td><b>1X(µL)</b><td><b>Master Mix(x16)(µL)</b> | ||
+ | <tr><td>Milli-Q H<sub>2</sub>O<td>41.8<td>668.6 | ||
+ | <tr><td>10x Pfu Buffer with MgSO<sub>4</sub><td>5<td>80 | ||
+ | <tr><td>dNTPs<td>1<td>16 | ||
+ | <tr><td>Forward Primer<td>0.5<td>8 | ||
+ | <tr><td>Reverse Primers<td>0.5<td>8 | ||
+ | <tr><td>Template DNA<td>1<td>16 | ||
+ | <tr><td>PFu polymerase<td>0.2<td>3.2 | ||
+ | </table><br> | ||
+ | |||
+ | <b>Objective:</b> Run PCR samples on a 2.5% agarose gel(1x TAE)for 70 minutes<br> | ||
+ | |||
+ | <table border ="3"> | ||
+ | <tr><td>lane<td><b>contents</b><td><b>Successful Ligation ?</b> | ||
+ | <tr><td>1<td>50bp ladder<td>--- | ||
+ | <tr><td>2<td>dt pSBIC3<td>--- | ||
+ | <tr><td>3<td>dt pTet<td>x | ||
+ | <tr><td>4<td>dt control<td>--- | ||
+ | <tr><td>5<td>sRBS-TetR<td>x | ||
+ | <tr><td>6<td>mRBS-TetR<td>? | ||
+ | <tr><td>7<td>TetR control<td>--- | ||
+ | <tr><td>8<td>pLacI-mRBS<td>x | ||
+ | <tr><td>9<td>pLacI-sRBS<td>? | ||
+ | <tr><td>10<td>pLacI control<td>--- | ||
+ | <tr><td>11<td>Mms6 pET-28a<td>no band | ||
+ | <tr><td>12<td>Mms6 control<td>--- | ||
+ | <tr><td>13<td>pBad-SRBS<td>x | ||
+ | <tr><td>14<td>pBad-mRBS<td>x | ||
+ | <tr><td>15<td>pBad control<td>--- | ||
+ | </table><br> | ||
+ | |||
+ | all ligations were transformed<br> | ||
+ | |||
+ | <b>Objective:</b> Transform<br> | ||
+ | |||
+ | <b>Method:</b> used [[Team:Lethbridge/Notebook/Protocols|Competent Cell Transformation]] protocol | ||
+ | * changes: | ||
+ | **used 50µL aliquottes of DH5 |
Revision as of 03:13, 26 August 2010
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Back to Lab Work
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
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
will PCR:
- ligations
- pBAD-mRBS
- pBAD-SRBS
- SRBS-tetR
- mRBS-TetR
- dt-pTet
- mms6-pET-28a
- dt-pSBIC3
- pLacI-SRBS
- controls
- pBAD
- TetR
- TetR
- pLacI
- Mms6
1X(µL) | Master Mix(x16)(µL) | |
Milli-Q H2O | 41.8 | 668.6 |
10x Pfu Buffer with MgSO4 | 5 | 80 |
dNTPs | 1 | 16 |
Forward Primer | 0.5 | 8 |
Reverse Primers | 0.5 | 8 |
Template DNA | 1 | 16 |
PFu polymerase | 0.2 | 3.2 |
Objective: Run PCR samples on a 2.5% agarose gel(1x TAE)for 70 minutes
lane | contents | Successful Ligation ? |
1 | 50bp ladder | --- |
2 | dt pSBIC3 | --- |
3 | dt pTet | x |
4 | dt control | --- |
5 | sRBS-TetR | x |
6 | mRBS-TetR | ? |
7 | TetR control | --- |
8 | pLacI-mRBS | x |
9 | pLacI-sRBS | ? |
10 | pLacI control | --- |
11 | Mms6 pET-28a | no band |
12 | Mms6 control | --- |
13 | pBad-SRBS | x |
14 | pBad-mRBS | x |
15 | pBad control | --- |
all ligations were transformed
Objective: Transform
Method: used Competent Cell Transformation protocol
- changes:
- used 50µL aliquottes of DH5