Barbara Lee Chow
blc123 at herald.usask.ca
Fri Jul 8 12:34:09 EST 1994
Hello out there. I'm kinda new to this e-mail thing so I hope this works!
I just completed my B.Sc. so I am a little bit new to this research thing.
I've been asked to arrest cells to obtain a synchronized population, release them, and take samples for RNA isolation.
I have been using alpha-factor to arrest the cells at the G1/S transition. The protocol I have been using calls for 2 treatments with 5 micrograms/ml alpha-factor each time, incubating the cells for 1.5 hr after each treatment.
I have a few questions which I hope some of you more experienced with these types of experiments can respond to:
1) Is there any other way(s) to achieve good arrest without using so much alpha-factor? Are 2 treatments really necessary, and if so, can the second treatment be <5 ug/ml?
2) I am using S. cerevisiae alpha-factor. Is S. kluyveri better/worse, or does it make any difference?
3) How important is the initial cell concentration? Most papers I've read say that the cells should be in early log phase, at a concentration anywhere from 2-5 x10e6.
4) Is there any way the cells can be manipulated to hinder their degradation of alpha-factor? I do have a bar1 mutant which is supersensitive, but was just wondering if there was any treatment that could be done on 'wild type' cells to achieve the desired effect
I know this request was a little lengthly, so thanks for listening. Any and all information is greatly appreciated!!
e-mail blc123 at herald.usask.ca
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