Team:Groningen/Notebook/16 July 2009

GVP Cluster
Colony PCR on cells containing the GVP and J23100 ligation product

- 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 - 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
 * Colonies are picked from the plate with a sterile toothpick and resuspended in 20 μL MQ
 * Mix (1):
 * Mix (2):


 * 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.

Vectors
ligation of constitutive promotors BBa_J23100, BBa_J23106, BBa_J23109 in vectors pSB1AC3 and pSB3K3.


 * cut for 15 min at 37&deg;, 10 min at 80&deg; 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&deg; 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.