Team:Groningen/Notebook/31 August 2009
From 2009.igem.org
(→Transporters) |
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'''Purification''' | '''Purification''' | ||
+ | |||
+ | [[Image:.jpg|240px]] [[Image:Generulers_1kb_marker_Fermentas.jpg]] | ||
+ | |||
+ | :→ From left to right: 1kB ladder, pSB1A2-pLacI-GVP (2x) ,Empty Slot, pSB1A2-pBAD-AraC-GVP (2x) , Empty Slot, pSB1AC3-H (2x) | ||
+ | |||
A "Agarose Gel DNA Extraction Kit" [http://www.roche-applied-science.com/pack-insert/1696505a.pdf Standard Protocol] from Roche Applied Science. | A "Agarose Gel DNA Extraction Kit" [http://www.roche-applied-science.com/pack-insert/1696505a.pdf Standard Protocol] from Roche Applied Science. | ||
:→ In step ten, 30μL MQ was added to the dry pellet and incubated at room temperature. | :→ In step ten, 30μL MQ was added to the dry pellet and incubated at room temperature. | ||
+ | |||
+ | |||
+ | '''Concentrations''' | ||
+ | |||
+ | {|cellpadding="2" cellspacing="1" border="4" | ||
+ | |'''Plasmid''' | ||
+ | |'''Conc. ng/μL''' | ||
+ | |'''260/280 | ||
+ | |'''260/230 ''' | ||
+ | |'''-20 box (michael | ||
+ | |'''Restriction Control''' | ||
+ | |- | ||
+ | |pLacI-GVP (EcoRI/PstI) | ||
+ | |5.0 | ||
+ | |1.47 | ||
+ | |1.04 | ||
+ | |? | ||
+ | |Yes (EcoRI/PstI) | ||
+ | |- | ||
+ | |pBad/araC-GVP (EcoRI/PstI) | ||
+ | |24.0 | ||
+ | |1.76 | ||
+ | |1.53 | ||
+ | |? | ||
+ | |Yes (EcoRI/PstI) | ||
+ | |- | ||
+ | |pSB1AC3 (EcoRI/PstI) | ||
+ | |24.4 | ||
+ | |1.77 | ||
+ | |1.69 | ||
+ | |? | ||
+ | |Yes (EcoRI/PstI) | ||
+ | |} | ||
+ | |||
+ | '''Ligation''' | ||
+ | |||
+ | A total amount of vector of 125-200ng was used (promoter-GVP) in a 1:2 and 1:3 ratio with insert. | ||
+ | |||
+ | (1:3) | ||
+ | :* 3 uL Ligase buffer | ||
+ | :* 1 ul T4 Ligase | ||
+ | :* 8 uL plasmid pSB1AC3 digested with EcoRI and PstI | ||
+ | :* 8 uL insert pBad-araC-GVP restricted with EcoRI and PstI | ||
+ | |||
+ | (1:2) | ||
+ | :* 3 uL Ligase buffer | ||
+ | :* 1 ul T4 Ligase | ||
+ | :* 11 uL plasmid pSB1AC3 digested with EcoRI and PstI | ||
+ | :* 5 uL insert pLac-GVP restricted with EcoRI and PstI | ||
+ | |||
+ | ''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 | ||
+ | |||
+ | |||
+ | :→ Positive control was BBa_J61002-J23101 plasmid, and negative control was MQ. | ||
===Transporters=== | ===Transporters=== |
Revision as of 11:53, 31 August 2009
Wet
GVP Cluster
Concentrations
Plasmid | Conc. ng/μL | 260/280 | 260/230 | -20 box (michael | Restriction Control |
pSB1A2-pLacI-GVP | 41.2 | 1.85 | 2.18 | ? | Yes (EcoRI/PstI) |
pSB1A2-pBad/araC-GVP | 421.7 | 1.84 | 2.34 | ? | Yes (EcoRI/PstI) |
pSB1AC3-H | 2.43.1 | 1.86 | 2.17 | All Used | Yes (Glycerol Stock) |
Restriction for Assembly
The vector pSB1A2 containing the pLacI/GVP and pBad/araC/GVP composite parts were cut with PstI and EcoRI to create correct ends for insert into pSB1AC3, which was also cut with EcoRI and PstI.
Plasmid | Amount μL | MQ μL | Fast digest buffer | EcoRI fast digest enzyme | XbaI fast digest enzyme | SpeI fast digest enzyme | PstI fast digest enzyme |
pSB1A2-pLacI-GVP | 15.0 | x | 3.0 | 1.0 | x | x | 1.0 |
pSB1A2-pBad/araC-GVP | 4.0 | 12.0 | 3.0 | 1.0 | x | x | 1.0 |
pSB1AC3-High | 6.0 | 9.0 | 3.0 | 1.0 | x | x | 1.0 |
Restriction was kept at 37C for 30 min. and put on ice until used for gel purification.
Purification
- → From left to right: 1kB ladder, pSB1A2-pLacI-GVP (2x) ,Empty Slot, pSB1A2-pBAD-AraC-GVP (2x) , Empty Slot, pSB1AC3-H (2x)
A "Agarose Gel DNA Extraction Kit" Standard Protocol from Roche Applied Science.
- → In step ten, 30μL MQ was added to the dry pellet and incubated at room temperature.
Concentrations
Plasmid | Conc. ng/μL | 260/280 | 260/230 | -20 box (michael | Restriction Control |
pLacI-GVP (EcoRI/PstI) | 5.0 | 1.47 | 1.04 | ? | Yes (EcoRI/PstI) |
pBad/araC-GVP (EcoRI/PstI) | 24.0 | 1.76 | 1.53 | ? | Yes (EcoRI/PstI) |
pSB1AC3 (EcoRI/PstI) | 24.4 | 1.77 | 1.69 | ? | Yes (EcoRI/PstI) |
Ligation
A total amount of vector of 125-200ng was used (promoter-GVP) in a 1:2 and 1:3 ratio with insert.
(1:3)
- 3 uL Ligase buffer
- 1 ul T4 Ligase
- 8 uL plasmid pSB1AC3 digested with EcoRI and PstI
- 8 uL insert pBad-araC-GVP restricted with EcoRI and PstI
(1:2)
- 3 uL Ligase buffer
- 1 ul T4 Ligase
- 11 uL plasmid pSB1AC3 digested with EcoRI and PstI
- 5 uL insert pLac-GVP restricted with EcoRI and PstI
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
- → Positive control was BBa_J61002-J23101 plasmid, and negative control was MQ.
Transporters
HmtA
The cloning strategy will be changed. First we will make a PCR2 and a PCR1.2 where F2-mut1rc plus Rev-mut2rc will generate a HmtA where we will add a prefix via a PCR with F1-REV. And than ready for cloning. But first we wait for a kit.
An aditional PCR started to get more megaprimer.
Metal Accumulation
Vectors
Dry
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