"
Team:Groningen/Notebook/16 July 2009
From 2009.igem.org
(Difference between revisions)
m (Initial dry log.) |
(→Vectors) |
||
| (13 intermediate revisions not shown) | |||
| Line 4: | Line 4: | ||
===GVP Cluster=== | ===GVP Cluster=== | ||
| + | |||
| + | '''Colony PCR on cells containing the GVP and J23100 ligation product''' | ||
| + | |||
| + | * Colonies are picked from the plate with a sterile toothpick and resuspended in 20 μL MQ | ||
| + | * Mix (1): | ||
| + | - 2.5 μL Taq buffer (NH4) | ||
| + | - 0.2 μL dNTP | ||
| + | - 2 μL MgCl | ||
| + | - 16.3 μL MQ | ||
| + | - 1 μL VF2 primer | ||
| + | - 1 μL VR primer | ||
| + | - 1 μL colony solution | ||
| + | - 1 μL Taq | ||
| + | * Mix (2): | ||
| + | - 2.5 μL Taq buffer (KCl) | ||
| + | - 0.2 μL dNTP | ||
| + | - 1.5 μL MgCl | ||
| + | - 16.8 μL MQ | ||
| + | - 1 μL VF2 primer | ||
| + | - 1 μL VR primer | ||
| + | - 1 μL colony solution | ||
| + | - 1 μL Taq | ||
| + | |||
| + | {| | ||
| + | ! PCR program | ||
| + | ! Temperature | ||
| + | ! Time | ||
| + | |- | ||
| + | |Denaturing | ||
| + | |95° | ||
| + | |5.00 min | ||
| + | |- | ||
| + | | | ||
| + | |Start Cycles 20X | ||
| + | |- | ||
| + | |Denaturing | ||
| + | |95° | ||
| + | |30 sec | ||
| + | |- | ||
| + | |Annealing | ||
| + | |55° | ||
| + | |30 sec | ||
| + | |- | ||
| + | |Elongation | ||
| + | |72° | ||
| + | |6.10 min | ||
| + | |- | ||
| + | | | ||
| + | |End cycles | ||
| + | |- | ||
| + | |Hold | ||
| + | |4° | ||
| + | |Forever | ||
| + | |} | ||
| + | |||
| + | * PCR products were analysed on gel (1% agarose, TBE, EtBr, 25 min, 100V) | ||
| + | |||
| + | Results: No bands | ||
| + | |||
| + | '''Inoculation of Colonies for restriction analysis''' | ||
| + | |||
| + | * 3 ml Ty (100 μg/ml Ampicillin) inoculated with colonies from plate (with sterile toothpick) | ||
| + | * Incubation o/n 37°C in shaker | ||
| + | |||
===Transporters=== | ===Transporters=== | ||
| + | |||
| + | We decided to make MasterMixes of our own. All but 1uL template, 2uL primers and 1uL taq for 25uL reactions. | ||
| + | {| | ||
| + | | | ||
| + | <!--Tabel 1 hier--> | ||
| + | |||
| + | {| border="1" | ||
| + | |+ '''1X MasterMix Buffer NH SO''' | ||
| + | ! Component | ||
| + | ! concentrations | ||
| + | ! volumes | ||
| + | |- | ||
| + | ! dNTP | ||
| + | | 0.2 mM | ||
| + | |8 uL | ||
| + | |- | ||
| + | ! MgCl | ||
| + | | 2.0 mM | ||
| + | |80 uL | ||
| + | |- | ||
| + | !taq buffer KCl | ||
| + | |1X | ||
| + | |100 uL | ||
| + | |- | ||
| + | ! MQ | ||
| + | | | ||
| + | |652 uL | ||
| + | |} | ||
| + | |width="10"| | ||
| + | | | ||
| + | <!--Tabel 2 hier--> | ||
| + | |||
| + | {| border="1" | ||
| + | |+ '''1X MasterMix Buffer KCl''' | ||
| + | ! Component | ||
| + | ! concentrations | ||
| + | ! volumes | ||
| + | |- | ||
| + | ! dNTP | ||
| + | | 0.2 mM | ||
| + | |8 uL | ||
| + | |- | ||
| + | ! MgCl | ||
| + | | 1.5 mM | ||
| + | |60 uL | ||
| + | |- | ||
| + | !taq buffer (NH)SO | ||
| + | |1X | ||
| + | |100 uL | ||
| + | |- | ||
| + | ! MQ | ||
| + | | | ||
| + | |672 uL | ||
| + | |} | ||
| + | |||
| + | |width="10"| | ||
| + | | | ||
| + | <!--Tabel 3 hier--> | ||
| + | |} | ||
| + | |||
| + | '''PCR''' | ||
| + | |||
| + | We need to have a product with the forward primer. All the other primes give a product. We wil make different PCR product which we can use as mega primers. Second, we will try different PCR programs for example reducing the annealing temperature. | ||
| + | |||
| + | <!--Tabel 2 hier-->{| border="1" {{table}} | ||
| + | |- | ||
| + | ! Component||B1||B2||C1||C2||A1||A2||E1||E2||F1||F2 | ||
| + | |- | ||
| + | ! MM | ||
| + | ||21||22||21||22||21||22||21||22||21||22 | ||
| + | |- | ||
| + | ! F 1uL | ||
| + | ||Fw||Fw||Fw||Fw||mut1||mut1||Rev||Rev||Fw||Fw | ||
| + | |- | ||
| + | ! R 1uL | ||
| + | ||Rev||Rev||mut2||mut2||PCR2||PCR2||mut1||mut1||mut1RC||mut1RC | ||
| + | |- | ||
| + | ! DNA 1uL | ||
| + | ||1||0||1||0||1||0||1||0||1||0 | ||
| + | |- | ||
| + | ! taq 1uL | ||
| + | ||1||1||1||1||1||1||1||1||1||1 | ||
| + | |} | ||
| + | |||
| + | {| {{table}} ! | ||
| + | !|'''PCR programs''' | ||
| + | !|'''B1''' | ||
| + | !|'''B2''' | ||
| + | !|'''C1''' | ||
| + | !|'''C2''' | ||
| + | !|'''A1''' | ||
| + | !|'''A2''' | ||
| + | !|'''E1''' | ||
| + | !|'''E2''' | ||
| + | !|'''F1''' | ||
| + | !|'''F2''' | ||
| + | |- | ||
| + | | Program||Temp||Time||Temp||Time||Temp||Time||Temp||Time||Temp||Time | ||
| + | |- | ||
| + | | hotstart||95||5 min||95||5 min||95||5 min||95||5 min||95||5 min | ||
| + | |- | ||
| + | | Touchdown||||||||||50 to 45||10X ||50 to 45||10X ||50 to 45||10X | ||
| + | |- | ||
| + | | Denaturing||||||||||95||30 sec||95||30 sec||95||30 sec | ||
| + | |- | ||
| + | | annealing||||||||||55||20 sec||55||20 sec||55||20 sec | ||
| + | |- | ||
| + | | elongations||||||||||72||60 sec||72||60 sec||72||60 sec | ||
| + | |- | ||
| + | | start cycles ##||30||X||30||X||25||X||25||X||25||X | ||
| + | |- | ||
| + | | Denaturing||95||30 sec||95||30 sec||95||30 sec||95||30 sec||95||30 sec | ||
| + | |- | ||
| + | | annealing||55||20 sec||55||20 sec||55||20 sec||55||20 sec||55||20 sec | ||
| + | |- | ||
| + | | elongations||72||150 sec||72||150 sec||72||60 sec||72||60 sec||72||60 sec | ||
| + | |- | ||
| + | | end cycles|||||||||||||||||||| | ||
| + | |- | ||
| + | | Final elongation||72||10 min||72||10 min||72||10 min||72||10 min||72||10 min | ||
| + | |- | ||
| + | | Hold||4||forever||4||forever||4||forever||4||forever||4||forever | ||
| + | |- | ||
| + | | | ||
| + | |} | ||
===Metal Accumulation=== | ===Metal Accumulation=== | ||
===Vectors=== | ===Vectors=== | ||
| + | |||
| + | ligation of constitutive promotors BBa_J23100, BBa_J23106, BBa_J23109 in vectors pSB1AC3 and pSB3K3. | ||
| + | |||
| + | {| | ||
| + | ! | ||
| + | !width="80" align="left"| pSB1AC3 | ||
| + | !width="80" align="left"| pSB3K3 | ||
| + | !width="130" align="left"| BBa_J123100 | ||
| + | !width="130" align="left"| BBa_J23106 | ||
| + | !width="130" align="left"| BBa_J23109 | ||
| + | |- | ||
| + | !align="left"|DNA | ||
| + | | 3ul | ||
| + | | 25ul | ||
| + | | 8,4ul | ||
| + | | 6ul | ||
| + | | 7,4ul | ||
| + | |- | ||
| + | !align="left"| Fast Digest Buffer | ||
| + | | 3ul | ||
| + | | 3ul | ||
| + | | 2ul | ||
| + | | 2ul | ||
| + | | 2ul | ||
| + | |- | ||
| + | !align="left"| EcoRI | ||
| + | | 2ul | ||
| + | | 2uL | ||
| + | | 1ul | ||
| + | | 1ul | ||
| + | | 1ul | ||
| + | |- | ||
| + | !align="left"| SpeI | ||
| + | | 2ul | ||
| + | | 2uL | ||
| + | | 1ul | ||
| + | | 1ul | ||
| + | | 1ul | ||
| + | |- | ||
| + | !align="left"| MQ | ||
| + | | 20ul | ||
| + | | | ||
| + | | 7,6ul | ||
| + | | 10ul | ||
| + | | 8,6ul | ||
| + | |} | ||
| + | |||
| + | * cut for 15 min at 37° , 10 min at 80° heat inactivation | ||
| + | |||
| + | ligation of pSB1AC3 and pSB3K3 with promotors | ||
| + | |||
| + | * 30ul reactions | ||
| + | :: 10ul cut vector | ||
| + | :: 10ul cut promotors | ||
| + | :: 2 ul T4 ligase | ||
| + | :: 3 ul 10x T4 ligation buffer | ||
| + | :: 5 ul MQ | ||
| + | |||
| + | * incubated at 4° overnight | ||
==Dry== | ==Dry== | ||
KB extended the Simbiology model to support OpG and OpH in addition to OpN. Jasper in the mean time added some more explanation to the modelling section on the [[Team:Groningen/Project/Accumulation|metal accumulation]] page. | KB extended the Simbiology model to support OpG and OpH in addition to OpN. Jasper in the mean time added some more explanation to the modelling section on the [[Team:Groningen/Project/Accumulation|metal accumulation]] page. | ||
| + | |||
| + | In the end of the afternoon we tried to find more modelling software. Likely candidates are SEMPPR (Japer) and cytoscape (KB) Furthermore KB is trying to get up to date with the paper knowledge. | ||
{{Team:Groningen/Notebook/Day/Footer}} | {{Team:Groningen/Notebook/Day/Footer}} | ||
Latest revision as of 15:14, 17 August 2009
[http://2009.igem.org/Team:Groningen http://2009.igem.org/wiki/images/f/f1/Igemhomelogo.png]
|
|---|
Wet
GVP Cluster
Colony PCR on cells containing the GVP and J23100 ligation product
- Colonies are picked from the plate with a sterile toothpick and resuspended in 20 μL MQ
- Mix (1):
- 2.5 μL Taq buffer (NH4) - 0.2 μL dNTP - 2 μL MgCl - 16.3 μL MQ - 1 μL VF2 primer - 1 μL VR primer - 1 μL colony solution - 1 μL Taq
- Mix (2):
- 2.5 μL Taq buffer (KCl) - 0.2 μL dNTP - 1.5 μL MgCl - 16.8 μL MQ - 1 μL VF2 primer - 1 μL VR primer - 1 μL colony solution - 1 μL Taq
| PCR program | Temperature | Time |
|---|---|---|
| Denaturing | 95° | 5.00 min |
| Start Cycles 20X | ||
| Denaturing | 95° | 30 sec |
| Annealing | 55° | 30 sec |
| Elongation | 72° | 6.10 min |
| End cycles | ||
| Hold | 4° | Forever |
- PCR products were analysed on gel (1% agarose, TBE, EtBr, 25 min, 100V)
Results: No bands
Inoculation of Colonies for restriction analysis
- 3 ml Ty (100 μg/ml Ampicillin) inoculated with colonies from plate (with sterile toothpick)
- Incubation o/n 37°C in shaker
Transporters
We decided to make MasterMixes of our own. All but 1uL template, 2uL primers and 1uL taq for 25uL reactions.
|
|
PCR
We need to have a product with the forward primer. All the other primes give a product. We wil make different PCR product which we can use as mega primers. Second, we will try different PCR programs for example reducing the annealing temperature.
| Component | B1 | B2 | C1 | C2 | A1 | A2 | E1 | E2 | F1 | F2 |
|---|---|---|---|---|---|---|---|---|---|---|
| MM | 21 | 22 | 21 | 22 | 21 | 22 | 21 | 22 | 21 | 22 |
| F 1uL | Fw | Fw | Fw | Fw | mut1 | mut1 | Rev | Rev | Fw | Fw |
| R 1uL | Rev | Rev | mut2 | mut2 | PCR2 | PCR2 | mut1 | mut1 | mut1RC | mut1RC |
| DNA 1uL | 1 | 0 | 1 | 0 | 1 | 0 | 1 | 0 | 1 | 0 |
| taq 1uL | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
| PCR programs | B1 | B2 | C1 | C2 | A1 | A2 | E1 | E2 | F1 | F2 |
|---|---|---|---|---|---|---|---|---|---|---|
| Program | Temp | Time | Temp | Time | Temp | Time | Temp | Time | Temp | Time |
| hotstart | 95 | 5 min | 95 | 5 min | 95 | 5 min | 95 | 5 min | 95 | 5 min |
| Touchdown | 50 to 45 | 10X | 50 to 45 | 10X | 50 to 45 | 10X | ||||
| Denaturing | 95 | 30 sec | 95 | 30 sec | 95 | 30 sec | ||||
| annealing | 55 | 20 sec | 55 | 20 sec | 55 | 20 sec | ||||
| elongations | 72 | 60 sec | 72 | 60 sec | 72 | 60 sec | ||||
| start cycles ## | 30 | X | 30 | X | 25 | X | 25 | X | 25 | X |
| Denaturing | 95 | 30 sec | 95 | 30 sec | 95 | 30 sec | 95 | 30 sec | 95 | 30 sec |
| annealing | 55 | 20 sec | 55 | 20 sec | 55 | 20 sec | 55 | 20 sec | 55 | 20 sec |
| elongations | 72 | 150 sec | 72 | 150 sec | 72 | 60 sec | 72 | 60 sec | 72 | 60 sec |
| end cycles | ||||||||||
| Final elongation | 72 | 10 min | 72 | 10 min | 72 | 10 min | 72 | 10 min | 72 | 10 min |
| Hold | 4 | forever | 4 | forever | 4 | forever | 4 | forever | 4 | forever |
Metal Accumulation
Vectors
ligation of constitutive promotors BBa_J23100, BBa_J23106, BBa_J23109 in vectors pSB1AC3 and pSB3K3.
| pSB1AC3 | pSB3K3 | BBa_J123100 | BBa_J23106 | BBa_J23109 | |
|---|---|---|---|---|---|
| DNA | 3ul | 25ul | 8,4ul | 6ul | 7,4ul |
| Fast Digest Buffer | 3ul | 3ul | 2ul | 2ul | 2ul |
| EcoRI | 2ul | 2uL | 1ul | 1ul | 1ul |
| SpeI | 2ul | 2uL | 1ul | 1ul | 1ul |
| MQ | 20ul | 7,6ul | 10ul | 8,6ul |
- cut for 15 min at 37° , 10 min at 80° heat inactivation
ligation of pSB1AC3 and pSB3K3 with promotors
- 30ul reactions
- 10ul cut vector
- 10ul cut promotors
- 2 ul T4 ligase
- 3 ul 10x T4 ligation buffer
- 5 ul MQ
- incubated at 4° overnight
Dry
KB extended the Simbiology model to support OpG and OpH in addition to OpN. Jasper in the mean time added some more explanation to the modelling section on the metal accumulation page.
In the end of the afternoon we tried to find more modelling software. Likely candidates are SEMPPR (Japer) and cytoscape (KB) Furthermore KB is trying to get up to date with the paper knowledge.
|
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
