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Team:Wageningen UR/Notebook/Proj2/May
From 2011.igem.org
May - Fungal Track 'n Trace
May 2
SOB medium, liquid and agar, were prepared and autoclaved today.
May 3
SOB agar was melted in a microwave and 12 plates were poured. To one plate kanamycin was added (final concentration 50 µg/ml). 1 litre minimal medium salts for Aspergillus medium were made at a 10x concentration. 2 liter complete medium was made and glucose was added to a final concentration of 50 mM. 15 grams of agar was added to one litre complete medium. All the comlete media were autoclaved. CCMB80 buffer was prepared, during pH adjustions the value increases to 7.6 and a slight yellow colour is formed. No precipitation is visible.
A DH5α strain containing a pAL85 plasmid (kanamycin resistance and pyrA complementation gene) was inoculated on the kanamycin plate. Top10 cells were plated on a regular SOB plate.
May 4
During the night the yellow colour of the CCMB80 buffer has precipitated and a dark brown cake has formed. The buffer is discarded and a new CCMB80 buffer is prepared. For the pH correction a lower molar concentration of KOH was used, so the pH did not increase past the critical point.
Complete medium agar was melted in the microwave. To 500 ml of liquidized CM agar, glucose was added using a 0.2 µm filter. Also 1 ml of vitamin solution was added over a 0.2 µm filter, from this complemented medium 7 big plates were poured. Additionally, some plates were prepared with sorbitol in stead of glucose.
A DH5α strain containing a pRLMex plasmid was inoculated on a SOB plate with ampicilin.
SOB medium was inoculated with Top10 and DH5α cells from the plates that were made yesterday.
May 5
DH5α, pHL85 and top10 were inoculated.
May 6
The concentration of Top10 cells in CCMB80 buffer was measured.
Cells containing DH5α and pAL85 plasmids were "miniprepped" using a Fermentas miniprep kit. This resulted in two eppendorf tubes, each containing 50 microliters of buffer with 39 nanograms of DNA per microliter.
May 10
The competence of the Top10 cells is measured.
Spore suspensions were made and put in a fridge.
Mes buffer, SMC buffer and STC buffer were prepared.
May 11
The number of colonies from yesterday's measurement of competence were counted. The cells are competent.
May 12
Four plates were inoculated with transformed Top10 cells (containing pDel1 and pDel2 plasmids) were put in a 37 degree (Celcius) stove overnight.
May 13
Lots of colonies grew on the plates that were prepared yesterday. The plates are put in a fridge.
May 16
Transformation medium is prepared.
May 17
We are making protoplasts. Thanks to Mark and a PhD student we now have two growth chambers for the fungal cells. In these growth chambers you have to grow the cells in liquid, which might be a problem. If all cells are just floating around, it will be hard to follow a signal through the hyphae.
