I am doing deletion..but the growth of cells is very slow and when I inoculate the colony into broth the growth is very less even after 24 hours. I want to do colony pcr... ll it helpful or not or suggest me some other method for deletion.
Hi Heena,
Can you provide a bit more information, please, it is difficult to give suggestions without any details as to what you are trying to do.
What kind of organism are you working with? Colony PCR can be very useful with some bacteria, like E.coli, but doesn't work well with others (Agrobacterium for instance). If the cells lyse well during the initial denaturation step of the PCR they can probably be used.
Colony PCR is usually used to detect whether your intended mutation/deletion is present, and is used because it is faster than doing full DNA extractions prior to PCR when you are screening many possible transformants. I have never heard of using it for making a mutant, only detecting it. What are you trying to delete, and what method are you using? If the gene is very important then growth will often be slow regardless of how it is done.
Alison
iam working with saccharomyces cerevisiae and I want to conform the deletion. Because of less no of cells or u can say growth I cannot proceed further for full DNA extraction method.so I think may be colony PCR will be helpful
Hi Heena,
Thanks for the details. I've never done colony PCR with S. cerevisiea so I can't say for sure if it will work, hopefully a yeast person will answer that. If not, yeast cells are tougher than E.coli, so you might want to boil a small amount of your sample for 10 minutes and then use that in your PCR, as that should release at least some DNA.
Good luck!
Alison
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