TUDelft/20 August 2008

=20th August 2008=

Testing the Primers (part 2)
Today another PCR was set up, again using the mastermix and composition also used for the first experiment (18th of August). Only this time the S. cerevisiae genes and cDNA were used. The cDNA contained 2ng/ul and the genes are: ERG8, ERG12, ERG13, HMG2 and MVD1. PCR program (with a 6ºC gradient this time) is as depicted below: 1. 5' @ 95ºC 2. 1' @ 95ºC 3. 1' @ 53.7ºC (gradient of 6C divided over 12 steps) 4. 1' @ 72ºC 5. repeat steps 2-4 29x (total of 30 cycles) 6. 5' @ 72ºC 7. forever @ 4ºC (PCR can be stopped and stored in the fridge at any time from this point on)

This time, the products were put directly on gel. The expected sizes are: ERG8 - ~1400bp, ERG12 - ~1300bp, ERG13 - ~1500bp, MVD1 - ~1200bp and HMG2 - ~3000bp. As there was no visible result on the ERGs gel, only the MVD1/HMG2 gel is shown:



Midiprep
All the plasmids from live stabs have been midiprepped. The gel showed multiple bands on pSB1A7, also the main band of this one was higher than the others. The rest looked OK. Concentrations of DNA were all between 40 - 100 ng/ul (total volume 500ul) with a 260/280 of 1.8 ± 0.1.