Greetings to science community,
I am operating Kluyveromyces marxianus yeast and want to generate haploid cells from diploid cells. However after 4 day since dissection, I found no growth of haploids. Could any one with similar experience shares your ideas with me?
Here is my protocol and observation:
1.) Yeast diploid cells were subcultured to OD600: 0.6, then transferred to sporulation medium of equivalent volume for spore generation.
*Sporulation medium recipe
https://cshprotocols.cshlp.org/content/2017/6/pdb.rec090076.short
2.) Start from the second day, around 20% tetras were observed under microscope.
3.) On the third and fourth days, tetra population did not change much but the shape were clearer (as photos appended).
4.) 50ul spore culture from 3rd and 4th day were spun down and transferred to 100ul 1M sorbitol.
5.) Zymolyase (5U/ul) from commercial gDNA extr. kit was diluted with 1M sorbitol 20 times and mixed with 10ul of step4. mixture.
6.) Incubate step5. at 30°C for 40min of zymolyase digestion reaction.
7.) Digestion mixture were dropped on YPD agar plate for tetra dissection.
8. After 4 days of incubation (30°C), no growth of haploids found.
With appreciation
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