Team:Groningen/Notebook/23 July 2009

From 2009.igem.org

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Wet

GVP Cluster

Transporters

Metal Accumulation

Vectors

  • Plasmid isolation from o/n cultures to gain more plasmid for comming cloning steps
    • Used Sigma plasmid isolation kit, eluted in 50ul MQ

DNA concentrations were:

Sample ng/ul 260/280 260/230
pSB3K3-high 7.8 1.94 1.47
pSB3K3-med 11.4 2.08 1.84
pSB3K3-low 6.8 1.72 1.44
pSB3K3 8.8 1.82 1.34
pSB1AC3-high 27.3 1.86 2.03
pSB1AC3-med 75.9 1.86 2.26
pSB1AC3-low 63.9 1.84 2.19
pSB1AC3 49.2 1.89 2.21
pSB1A2-pLac 1 18.5 1.78 2.00
pSB1A2-pLac 2 21.7 1.83 1.93
pSB1A2-K0077124 (used as + control for PCR) 48.0 1.85 2.09
  • Gradient PCR with VR/VF2 on positive controls: pSB3K3, pSB1A2-K077124 and negative controls: pUC and MQ
VR-VF2
Component amount
MasterMix NH4 21 uL
VF2 primer 1 uL
VR primer 1 uL
DNA 1 uL
Taq polymerase 1 uL
DNA and primers
DNA cup nr 1 2 3 4 cup nr 5 6 7 8
A BBa_K077124, in pSB1A2, VR/VF2 iGEM09 VR/VF2 iGEM08Aug
B pSB3K3 VR/VF2 iGEM09 VR/VF2 iGEM08Aug
C Blanco VR/VF2 iGEM09 VR/VF2 iGEM08Aug
D pUC VR/VF2 iGEM09 VR/VF2 iGEM08Aug


VR VF2 gradient program
Stage Temperature Time
Denaturing 95° 4 min
Start Cycles 30X
Denaturing 95° 30 sec
Annealing Gradient 51°-60° 30 sec
Elongation 72° 1.50 min
End cycles
Final elongation 72° 10 min
Hold Forever
Also pSB1A2-K077124 will be cut by EcoRI and SpeI (as done on 22 July) to check the construct. The expected fragments are: 1563bp (insert specific) and 2056bp (vector).

No colonies, also after another o/n @ 37dg. So the transformation has to be redone, and as the ORI may be induced by IPTG, the transformants will be plated on LBA-Kan-IPTG then.

  • O/n culture of single colonies
    • pSB1A2-R0010 (pLac) (from 2 different transformant, 1 single colony) in LB-Amp
    • pSB1AC3 with 3 promoters in LB-Cam
    • pSB3K3 with 3 promoters in LB-Kan
  • PCR on pSB1A2-R0010 (pLac), pSB1AC3 with 3 promoters,pSB3K3 with 3 promoters and the negative controls: pSB3K3 and pSB1AC3.

Pipeting scheme as done on 21 July on pSB3K3, but with a change in the PCR program: elongation time of 30sec and a Tm of 54dg. Expected fragments will be:

    • pSB1AC3 with 3 promoters - 350bp
    • pSB3K3 with 3 promoters - 350bp
    • pSB3K3 and pSB1AC3 - 316bp
    • pSB1A2-R0010 (pLac) - 438bp

Dry

As Nienke's preliminary RPU computations looked promising Jasper started to redo them using more data, and with the goal to ultimately get the results on the Wiki.

KB put the most essential equations of ArsB on the wiki. The afternoon was spent studing more closely the experimental data of the [paper] found yesterday.


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