PCR colony screening - (Apr/20/2005 )
hi, I want to try making a screening of bacteria colony direcly by PCR.
Someone have just tryed this screening procedure?
I hope it works well also because minipreparation of DNA for colony screening is long and expensive...
If someone has some other ideas for a fast screening, please let me know.
Thank for all idea or suggestion
I commonly use bacteria colony PCR for screening for those reasons exactly.
I innoculate a PCR mix of water, 10 x buffer and dNTPs with a bacterial colony (don't forget to also innoculate some broth so you don't lose the colony). Heat this PCR mix for 10 minutes at 100 degrees celcius and allow to cool to room temp (not everyone does this but I find it helps). Add primers and Taq and do a 30 cycle PCR.
The best way is to use a primer within the vector (I commonly use M13F) and an internal fwd/rev primer. If this is not practical you can use your specific PCR primers for your insert.
Hope this helps if you want any more details give us an email.
I DO NOT UNDERSTAND THE FUNDA BEHIND THE COLONY PCR, how will DNA come out ? yes this is known that cell wall lyses on coming in contact with water . can u explain me the mechanism behind it?
Colony PCR is a quick and relatively cheap technique to identify which clones have taken up your plasmid of interest, without having to miniprep. The fundamentals are quite simple; you transform your plasmid into your e. coli and grow on selective plate overnight. You then pick a single colony and innoculate some broth (so you don't lose the colony) and also a PCR mix or sterile water. This will lyse the bacteria so that tube will contain a small amount of vector DNA (released from the cytosol). Using this as your template you can PCR amplify the gene of interest to determine its presence or absence. You can then go back to the culture you set up and grow overnight for miniprep. Significantly cuts down the number of minipreps especially where you can't do blue/white colony screening.
It is especially helpful if you have a non-directional ligation, or your background ligation still gave you a fair number of colonies (when there shouldn't be any).
Because in both cases you're odds of getting the right plasmid construct is significantly reduced.
thanx for explaining in detail. it worked out
Hi, I have a question, can I use colony pcr to detect my gene that recombinate into the chromosome? Thanks in advance
Yes provide that you can design primers that are specific to your recombination event.
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Can you use colony PCR with B. subtilis or polymerases other than taq (like pfx/pfu)?
I currently have a ton of money but not a lot of time, so I might select this technique if it seems sensible.
Any DNA polymerase will be fine. I have never performed colony PCR on Bacillus but I can't imagine that it would not lyse if heated to 100C for 5 minutes.
I generally don't use the colony directly in the PCR, but use a separate PCR tube with 20 µl of 10/1 TE + 1% Triton and use 2 µl of this lysate in my PCR after heating to 100C for 5 minutes. The advantage of this approach is you can store the DNA as well as perform more than 1 PCR.
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