Team:Groningen/Notebook/12 September 2009

GVP Cluster
Plates

The ampicillin stocks showed signs of failure (growth on negative control, overgrowth of low concentration plates, etc.), and therefor a new batch was made and used for agar plates to check if the ampicillin works. A positive and negative transformation with the new competent cells was performed and plated on LB-agar-amp100 plates in low and high concentration.


 * → The plates showed the expected amount of colonies, and also red in the case of the positive plates. The three colonies on the negative plate can be taken into acount when plating transformed cells.


 * → The two plates with stripes of pZntR-GVP (pSB2K3) and pCueO-GVP (pSB2K3) colonies showed growth and some single colonies.


 * → All six plates were stored in the fridge for further use on monday.

Cultures

The overnight cultures with LB-amp100 medium of colonies E.coli TOP10 with pLacI-GVP in pSB1A2 (4x) and the positive and negative control plates, showed no growth.


 * → The lack of growth can be caused by the failing ampicillin stocks. The cells have probably lost their plasmid during growth on agar plates after transformation, due to the lack of need for ampicillin resistance. The colonies taken from the plates did not grow on the new ampicillin batch, and is a second conformation of our suspicions.

The overnight cultures with LB-kan50 medium of colonies E.coli TOP10 with pZntR-GVP and pCueO-GVP in pSB2K3 (two of each) showed growth.


 * → From each culture 5mL was made into a pellet and stored in the -20 box of Michael. The pellets will be used for plasmid isolation on monday, and restriction to check if the GVP with promoter (~6000bp) and the vector (~4400bp) are as expected (self ligation would place the GVP construct in a ~3000bp vector.