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Team:Groningen/Notebook/10 August 2009
From 2009.igem.org
(Difference between revisions)
(→Metal Accumulation) |
m (→GVP Cluster) |
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|pSB1AC3-H SpeI/PstI | |pSB1AC3-H SpeI/PstI | ||
| - | | | + | |17. |
|? | |? | ||
|? | |? | ||
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|pSB1AC3-M SpeI/PstI | |pSB1AC3-M SpeI/PstI | ||
| - | | | + | |20. |
|? | |? | ||
|? | |? | ||
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|pSB1AC3-L SpeI/PstI | |pSB1AC3-L SpeI/PstI | ||
| - | | | + | |17. |
|? | |? | ||
|? | |? | ||
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|GVP XbaI/PstI restricted | |GVP XbaI/PstI restricted | ||
| - | | | + | |12.3 |
|? | |? | ||
|? | |? | ||
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''Incubate:'' | ''Incubate:'' | ||
:* 30 min @ ice | :* 30 min @ ice | ||
| - | :* | + | :* 90 sec 42°C |
:* 2 min @ ice | :* 2 min @ ice | ||
:* add 800uL LB-medium | :* add 800uL LB-medium | ||
:* incubate for 1 h at 37°C | :* incubate for 1 h at 37°C | ||
| - | :* plate on LB-amp<sub>50</sub> plates | + | :* plate on LB-amp<sub>50</sub>/chlo<sub>20</sub> plates |
'''Over Night Cultures''' | '''Over Night Cultures''' | ||
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Over night cultures in 4mL LB-amp<sub>100</sub> medium were prepared from the following glycerol stock: | Over night cultures in 4mL LB-amp<sub>100</sub> medium were prepared from the following glycerol stock: | ||
| - | :→ E.coli TOP10 high const. promoter | + | :→ E.coli TOP10 high const. promoter (-80 no.12) |
| - | :→ E.coli TOP10 med. const. promoter | + | :→ E.coli TOP10 med. const. promoter (-80 no.11) |
| - | :→ E.coli TOP10 low const. promoter | + | :→ E.coli TOP10 low const. promoter (-80 no.10) |
and put in the 37°C waterbath at 200 rpm. | and put in the 37°C waterbath at 200 rpm. | ||
Revision as of 15:11, 10 August 2009
[http://2009.igem.org/Team:Groningen http://2009.igem.org/wiki/images/f/f1/Igemhomelogo.png]
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Wet
GVP Cluster
Restriction for Assembly
The vector [http://partsregistry.org/wiki/index.php?title=Part:pSB1AC3 pSB1AC3] containing the [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 high], [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23106 medium] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23109 low] constitutive promoters were cut with PstI and SpeI to create correct ends for insert of GVP biobrick [http://partsregistry.org/wiki/index.php?title=Part:BBa_I750016 BBa_I750016], which was cut with XbaI and PstI.
- 3 to 10μL plasmid in MQ (1.0μg)
- 6 to 13μL MQ (end volume of 20μL)
- 2μL Fast digest buffer
- 1μL PstI fast digest enzyme
- 1μL SpeI/XbaI fast digest enzyme
Restriction was kept at 37C for 30 min. and put on ice until used for gel purification.
Purification
Concentrations
| Plasmid | Conc. ng/μL | 260/280 | 260/230 | -20 box (michael | Restriction Control |
| pSB1AC3-H SpeI/PstI | 17. | ? | ? | ? | ? |
| pSB1AC3-M SpeI/PstI | 20. | ? | ? | ? | ? |
| pSB1AC3-L SpeI/PstI | 17. | ? | ? | ? | ? |
| GVP XbaI/PstI restricted | 12.3 | ? | ? | ? | Gel |
Ligation
(1:3)
- 4 uL Ligase buffer
- 2 ul T4 Ligase
- 8 uL plasmid pSB1AC3-L digested with PstI and SpeI
- 8 uL insert GVP restricted with XbaI and PstI
(1:6)
- 4 uL Ligase buffer
- 2 ul T4 Ligase
- 8 uL plasmid pSB1AC3-M digested with PstI and SpeI
- 4 uL insert GVP restricted with XbaI and PstI
Incubate:
- 25°C 50min.
- kept on ice for 10min.
Tranformation
- add 10uL of the pSB1AC3-GVP ligation product to 50uL competent E.coli TOP10 cells.
Incubate:
- 30 min @ ice
- 90 sec 42°C
- 2 min @ ice
- add 800uL LB-medium
- incubate for 1 h at 37°C
- plate on LB-amp50/chlo20 plates
Over Night Cultures
Over night cultures in 4mL LB-amp100 medium were prepared from the following glycerol stock:
- → E.coli TOP10 high const. promoter (-80 no.12)
- → E.coli TOP10 med. const. promoter (-80 no.11)
- → E.coli TOP10 low const. promoter (-80 no.10)
and put in the 37°C waterbath at 200 rpm.
Transporters
Metal Accumulation
- Check colonies of E. coli + pGB68 (mymT)
- There were colonies on the MymT plates (a lot on the + control, but also 2 on - control...), 4 colonies were picked for o/n culture and colony PCR.
- Colony PCR as done for pSB1AC3-HML+RFP and pSB3K3-HML+RFP though the PCR program was slightly modified:
- Initial denat 95°C for 5min
- Touchdown 10x
- denat 95°C for 30sec
- anneal 62-50°C for 30sec
- elong 72°C for 30sec
- Cycles 20x
- denat 95°C for 30sec
- anneal 58°C for 30sec
- elong 72°C for 30sec
- Final elong 72°C for 5min
- Hold 4°C infinite
- Run on 1%Agarose gel.
- Contents: Marker-MymT(colony)1-MymT2-MymT3-MymT4
- On the gel a very faint band may be seen at the lanes with MymT3 and MymT4.. But there are too faint to be sure about there nature, it may also be possible that there unspecific products
- Amplify SmtA and SmtA-GST by PCR
- Use protocol as used for amplification of SmtA and SmtA-GST written on 7 August 2009.
- PCR program was modified to get the following:
- Initial denat 95°C for 5min
- Touchdown 10x
- denat 98°C for 30sec
- anneal 65-58°C for 1min:30sec
- elong 72°C for 1min:20sec
- Cycles 25x
- denat 98°C for 30sec
- anneal 59°C for 1min:30sec
- elong 72°C for 30sec
- Final elong 72°C for 5min
- Hold 4°C infinite
- Run on 1%Agarose gel.
Vectors
Dry
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