hey all
i need to extract DNA from yeast cells. but how can i do it from one yeast or very low number of yeast cells. is there a special protocol for it?
Thanks
May
Dear May
Could you please describe your problem a bit more?Why do you work with so few cells?
In what physical environment (flat surface,bulk)?Do you need double strands or single strands suffice?
Can you increase the culture volume? That's probably the easiest, most straightforward way to increase the number of cell and therefore the quantity of DNA.
Apart from that, certain protocoles are more efficacious than other at extracting DNA...
This article might be of interest:
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0115960
I'm guessing you're doing nanopore sequencing? Have you cultured the yeasts or are they environmental? If it's just for PCR, I would do a dilution plate series, then freeze the suspensions. When you've estimated the number of (viable) yeast cells present in a given volume, eg. the 5th dilution may have 500 cells per mL, you could then use 2 uL directly as a template for PCR. You would need to do a number of reactions to ensure some have 1 cell as others will have more or less. There will also be a number of non-viable cells that may act as templates. If you have different downstream applications that actually require DNA extraction, you could centrifuge a given volume estimated to give 1 cell (from the dilution series), remove the supernatant and boil for a short time in a small volume of chelex. Best of luck!
WOW, thank you all the the quick replays.
i will explain a bit:
i am applying Detergent incubation on the yeast cell and and after that the cell isn't alive anymore so i can no increase the culture volume. i want to see a DNA sequence from a specific yeast cell, and i was wondering if it possible is there a machine that connects to the FACS immediately and can give me a DNA sequence of a single cell?
Just to confirm... are you chasing the genome sequence of the cell, or a given target sequence (conventional or quantitative PCR)? If the latter, you shouldn't have too many problems. If the former, I agree with Mathew: very difficult!
anthony young- i have a plasmid inside that i want to reach and aeequence.. is it the first or the latter? :)
can i take a single cell and extract the DNA and sequence it? then i will have the sequence and i could enter it again to the yeast and express my engineered protein...
If your yeast cell has a plasmid, you could follow Simon's suggestion above and bulk it up in culture. Then you could do a mini-prep to purify the plasmid. I guess the other thing to bear in mind is that there are multiple pathways to any given objective, and if you wish to express an engineered protein, there may be simpler ways.
Hmmm... I'm not sure I understand the question you're trying to answer, but is there no chance you could bulk your yeast before the detergent incubation?? Otherwise, if you want to get the DNA sequence from a single dead cell you may wish to look into some of the amplification techniques applied in archaeology or some nanopore-based technique. Sorry I can't help ):
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology