We used Promega GoTaq® Green Master Mix to check the targeted deletion of genes from the genome of fission yeast, S. pombe. We tested the targeted disruption of ase1+, encoding a conserved microtubule-bundling protein(1)
. GoTaq® Green Master Mix is a premixed, ready-to-use solution containing GoTaq® DNA Polymerase, dNTPs, MgCl2, reaction buffers and gel loading solution.
Materials and Methods
An S. pombe strain was transformed with a DNA fragment carrying the kanMX6 cassette, conferring G-418 resistance, flanked by ase1 target sequences(1)
. Colonies resistant to G-418 were screened by PCR for correct disruption of the ase1+ gene.
S. pombe strains were maintained in YE medium(3)
. The oligonucleotide primers designed to check the disruption of ase1+ were obtained from IDT DNA technology (Coralville, IA) and are as follows:
Ase1 5´FL: 5´ ttactttcgcaggaaaaggc 3´
KanMXRev: 5´ ccggcgcaggaacactgcc 3´
We tested reactions in a final volume of either 10µl or 20µl. In our experience, the 20µl reactions worked best. For each primer, 0.5µl of a 20µM stock was added to each reaction and was diluted to 10µl with water. To this primer mix, 10µl of GoTaq® Green Master Mix was added to have a final volume of 20µl per reaction. A toothpick of cells was added to the reactions, and PCR was performed under the following conditions: 95°C for 5 minutes; 12 cycles of 95°C for 45 seconds, 60°C for 1 minute and 72°C for 2.5 minutes; 25 cycles of 95°C for 45 seconds, 52°C for 45 seconds and 72°C for 2.5 minutes. The reaction products were separated on a 1% agarose gel and visualized.
The results of the PCR are shown in Figure 1. Lanes 1, 2 and 6 contain a PCR product of ~800bp representing the disruption of ase1+ with kanMX6. Lanes 9 and 10 represent positive and negative control for the disruption, respectively. The low molecular weight bands presumably represent excess primers and/or denatured DNA from the reactions. As shown in Figure 1, the GoTaq® Green Master Mix is very efficient in the amplification of DNA fragments from yeast colonies. In summary, GoTaq® Green Master Mix is easy to use, reliable, versatile and efficient in the generation of PCR products from yeast colonies.