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Team:Groningen/Notebook/21 September 2009
From 2009.igem.org
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===GVP Cluster=== | ===GVP Cluster=== | ||
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'''Planning''' | '''Planning''' | ||
:→ {{todo}} work out the wiki page for GVP | :→ {{todo}} work out the wiki page for GVP | ||
| + | ::* made a layout | ||
| + | ::* still have to read all articles | ||
| + | ::* modeling will stay as it is, has been done by modeling people | ||
:→ {{done}} make a doodle for presentation planning (1-19 oct.) | :→ {{done}} make a doodle for presentation planning (1-19 oct.) | ||
| - | :→ {{ | + | :→ {{done}} media attention |
| + | ::* mail to UK, Ing., St. Gen. | ||
| + | ::* facebook account with link to twitter | ||
| + | ::* ethics survey link on facebook and twitter | ||
:→ {{done}} place an ethics survey link on twitter | :→ {{done}} place an ethics survey link on twitter | ||
| - | :→ {{ | + | :→ {{done}} clone pArsR-GVP into pSB2K3 |
| + | ::* {{todo}} still need to make glycerol stock | ||
:→ {{todo}} clone repeat out of GVP cluster | :→ {{todo}} clone repeat out of GVP cluster | ||
:→ {{todo}} make glycerol stocks of constructs | :→ {{todo}} make glycerol stocks of constructs | ||
| Line 39: | Line 45: | ||
|1.0 | |1.0 | ||
|} | |} | ||
| + | |||
| + | |||
| + | '''Purification''' | ||
| + | |||
| + | [[Image:Zymoclean Gel DNA Recovery Kit (D4001) 2.jpg|thumb|150px| www.zymoresearch.com]] | ||
| + | |||
| + | |||
| + | |||
| + | :→ In step 7 the fragments were eluted in 12μL MQ and was stored on ice until use. | ||
| + | |||
| + | :→ The concentration of GVP fragment with X/P sticky ends was 81.9 ng/μL, and was stored in fridge for restriction by MvaI/XhoI on Tuesday. | ||
| + | |||
| + | |||
| + | '''Ligation''' | ||
| + | |||
| + | A total amount of vector of 100ng was used in a 1:3 ratio with insert. | ||
| + | |||
| + | :* 3 uL Ligase buffer | ||
| + | :* 1 uL T4 Ligase | ||
| + | :* 3 uL GVP fragment 3300bp | ||
| + | :* 3 uL GVP fragment 2200bp | ||
| + | :* 4 uL plasmid J61035 XbaI/PstI | ||
| + | :* 8 uL GVP-insert (restricted with MvaI/XhoI) | ||
| + | |||
| + | |||
| + | ''Incubate:'' | ||
| + | :* 25°C 50min. | ||
| + | :* kept on ice for 10min. | ||
| + | |||
| + | '''Tranformation''' | ||
| + | :* add 10uL of the 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-amp<sub>100</sub> plates | ||
| + | |||
| + | |||
| + | :→ Negative control was MQ and positive was J61002-J23101. | ||
===Transporters=== | ===Transporters=== | ||
Revision as of 12:09, 21 September 2009
[http://2009.igem.org/Team:Groningen http://2009.igem.org/wiki/images/f/f1/Igemhomelogo.png]
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Wet
GVP Cluster
Planning
- → TODO work out the wiki page for GVP
- made a layout
- still have to read all articles
- modeling will stay as it is, has been done by modeling people
- → DONE make a doodle for presentation planning (1-19 oct.)
- → DONE media attention
- mail to UK, Ing., St. Gen.
- facebook account with link to twitter
- ethics survey link on facebook and twitter
- → DONE place an ethics survey link on twitter
- → DONE clone pArsR-GVP into pSB2K3
- TODO still need to make glycerol stock
- → TODO clone repeat out of GVP cluster
- → TODO make glycerol stocks of constructs
- → TODO enter info on part registry
Restriction for Assembly
The plasmid of pMA-gvpL insert was cut with MvaI/XhoI to cut out the entire part of wanted fragment.
| Plasmid | Amount μL | MQ μL | R-digest buffer | MvaI digest enzyme | XhoI digest enzyme |
| pArsR-GVP no.1 | 10.0 | 6.0 | 3.0 | 1.0 | 1.0 |
Purification
_2.jpg)
- → In step 7 the fragments were eluted in 12μL MQ and was stored on ice until use.
- → The concentration of GVP fragment with X/P sticky ends was 81.9 ng/μL, and was stored in fridge for restriction by MvaI/XhoI on Tuesday.
Ligation
A total amount of vector of 100ng was used in a 1:3 ratio with insert.
- 3 uL Ligase buffer
- 1 uL T4 Ligase
- 3 uL GVP fragment 3300bp
- 3 uL GVP fragment 2200bp
- 4 uL plasmid J61035 XbaI/PstI
- 8 uL GVP-insert (restricted with MvaI/XhoI)
Incubate:
- 25°C 50min.
- kept on ice for 10min.
Tranformation
- add 10uL of the 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-amp100 plates
- → Negative control was MQ and positive was J61002-J23101.
Transporters
HmtA
Nieuwe poging om HmtA te krijgen. Via verschillende routes, PCR1 met F1 mut1rc en EcoRI cut pBAD met 742 f2 en PCR1 & 2.
Metal Accumulation
Vectors
Dry
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