Latest revision as of 08:52, 10 September 2009

Project progress

FACS measurements:
- The cultures from shift 2 had similar results as shift one. However, the GFP signal measured from the LigA construct was stronger. Thus, cells definitely need to grow before undergoing irradiation.
The dilutions and the shift one cultures put overnight did not show any significant results.
was miniprepped and nanodropped

Part	concentration (ng/μl)	260/280 λ
(A)	238,9	1,95
(B)	248,8	1,91

After nanodropping, was restricted with SpeI and PstI, while was cut with PstI and XbaI.
A gel electrophoresis and extraction was performed. This showed an unexpected 800 bp signal at the lanes with . This is in fact due to the vector in which this part is ligated. contains a reporter gene, RFP. Thus, in combination with the promotor, a construct is formed that can be used to measure activity of this promotor. Since the promotor is flanked by standard assembly restriction sites we can replace the current promotor () by any other promotor, for instance .
DB3.1, and LigA were ented in liquid culture and grown overnight to make glycerol stocks and competent cells

The four grown colonies are put on liquid culture
New (again...) restriction digest on sam5, sam8, fcs and ech, genes were miniprepped on Tuesday.
- Overnight at 16°C
The plates with sam5, sam8, ech and fcs were re-ented. From sam5 and fcs four colonies were picked and plated.

The PCR from yesterday did not deliver any results, probably because of an error when preparing the PCR mix. A new PCR was prepared and started today.

there was made a controllgel of virA for our basicproduct, purification and nanodrop
the two ligations X+K were checked and electroporated
X was digested again and ligated in K
There were fluid cultures made for ligation of Y+K
A new PCR was done for rpoA mutagenesis : elongationstep was now taken 4 minutes instead of 1 minute

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