It is better to go for fresh cells. some time degraded DNA may result in non specific amplification. If cells are stored under low temperate can be used directly.
If your primers are very specific you can try to perform your PCR directly on dead bacteria, but as said Ganapati Bhat, you can also use low temperature stored bacteria. You can store bacterial colonies more than 4 weeks.
Good Luck
In principle colony PCR on dead bacteria should work; but it depends mainly on the "cause of death" and the subsequent storage. It would be great to know this in your case and why you want to do colony PCR with dead bacteria; do you want to check if a inserted plasmid/element is lethal when present? If your downstream application is molecular cloning or protein overexpression, I´d not recommend to do work with dead bacteria and I´d absolutely not recommend storing bacteria colonies in fridges for longer than absolutely necessary.
Please report whether your PCR worked or not :-)
Previously I have done colony PCR on my clones, they all showed positive results. After some time, I did the same colony PCR on them but they are not showing any result, even I tried to subculture again. May I know the reason? I seriously need some helps!
1. Be careful, it can be just a problem of PCR, sometimes you have to change some parameters depending on the material you are using!!
2. If you stored your clones on Room Temperature a long time (more than 2 weeks) or at 4 degrees more than 4 weeks, it s normal that you have nothing : degraded DNA may result in non specific amplification, so you got negative results
3. I know that yeast can reject the vector after few days in non selective medium, and sometimes on selective one also!! Bacteria need more long time to reject plasmids, but it depend on the genes and selection you are working with
Good luck
As it looks, your plasmids are most likely lost if you have not made any glycerol stocks. Even though most of the times colony PCR is fine to check clones, I would recommend doing minipreps as well and to do restriction-digestion analysis. This will tell you if you still have your plasmid, if it´s lost completely or if it has recombined.
What bacterial strain where you using? Overexpression strains are absolutely NOT able to keep plasmids intact for a longer time; they are engineered to grow well and produce a lot of protein so they lack several mutations (e.g. in a recombinase) that library-strains such as DH5alpha have and therefore will recombine your plasmid if they get the chance to do so. THerefore, it´s often recommended to transform overexpression strains fresh every time; or to at least make glycerol stocks ASAP and always start work with bacs from the glycerol stock.
Depending on the chances of getting good clones from a ligation I either make glycerol stocks directly from transformants and discard those that are not good. For this I pick clones with a 200 uL tip, seed a 5 mL culture incubate o/n, the next day I use part of the culture for the glycerole stock the rest I use for plasmid preparation and restriction analysis. In addition one can use the rest of the colony on the 200 uL tip to make colony PCR. If chances are low to have good clones and many clones need to be screened, I´d pick clones for the colony PCR as follows: take the colony with a 200 uL tip, streak on a small sector of a fresh plate as a "backup copy" and use the rest of the colony on the tip to do the colony PCR. Then as soon as the good clones are identified I make glycerol stocks as described above (including plasmid prep and restiction digestion analysis).
I ALWAYS sequence inserts to make sure there are no point-mutations or other artefacts introduced during cloning.
So I guess most of what I could provide is advice for future experiments :-/
Basically I have huge number of environmental isolate preserved in glycerol (15%). can I use these sample direct for PCR amplification 16S rDNA gene. and what will the procedure. please guide meLooking forward for your response.ThanksRafiq
Hi dear all,
please any of you know about the PCR from the glycerol stock.
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