Team:Groningen/Notebook/30 July 2009

Transporters
GlpF


 * Redone PCR3

The resulting PCR product was purified using a high pure PCR product purification kit (Roche). concentration was measured using Nanodrop;

Restiction was done on the GlpF PCR3 (30/07) with XbaI and PstI as noted on the notebook of 28/07/09 The resticted GlpF PCR3 (30/07) was ligated with psB1AC3 resticted with EcoRI and SpeI.


 * Ligation mix:

Vectors
Results were disappointing, no colonies on plate. Hope the fresh ligase and ligase buffer (10x) will help the new ligation that was started. New vectors were cut and purified from gel. 

In a total volume of 20uL, 2uL 10x Ligase Buffer and 1uL T4 DNA Ligase.
 * Ligation (ratio 1:3) of pSB1AC3-HML and pSB3K3-HML + RFP and pSB3K3 and pSB1AC3 + pLac
 * Calculated the volumes of vector and insert with Cloning Tools 6.0, using:
 * Amount: 200ng
 * Excess insert: 3x
 * The fragments were ligated using PCR program AC3 ligation:
 * Use 5uL ligation mixture for transformation.
 * Transform E. coli TOP10 via normal protocol.

Dry
We worked on migrating graphs from Google spreadsheets to Wiki tables, making sure that at least the raw data used to generate the graphs is "safe".