Team:Groningen/Notebook/16 July 2009

From 2009.igem.org

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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 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.

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
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

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.

ComponentB1B2C1C2A1A2E1E2F1F2
MM 21222122212221222122
F 1uL FwFwFwFwmut1mut1RevRevFwFw
R 1uL RevRevmut2mut2PCR2PCR2mut1mut1mut1RCmut1RC
DNA 1uL 1010101010
taq 1uL 1111111111
PCR programs B1 B2 C1 C2 A1 A2 E1 E2 F1 F2
ProgramTempTimeTempTimeTempTimeTempTimeTempTime
hotstart955 min955 min955 min955 min955 min
Touchdown50 to 4510X 50 to 4510X 50 to 4510X
Denaturing9530 sec9530 sec9530 sec
annealing5520 sec5520 sec5520 sec
elongations7260 sec7260 sec7260 sec
start cycles ##30X30X25X25X25X
Denaturing9530 sec9530 sec9530 sec9530 sec9530 sec
annealing5520 sec5520 sec5520 sec5520 sec5520 sec
elongations72150 sec72150 sec7260 sec7260 sec7260 sec
end cycles
Final elongation7210 min7210 min7210 min7210 min7210 min
Hold4forever4forever4forever4forever4forever

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.


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