- BioForum
- → Viewing Profile: Posts: Akdor
Find contentCommunity Stats
- Group Active Members
- Active Posts 22
- Profile Views 401
- Member Title member
- Age Age Unknown
- Birthday Birthday Unknown
-
Gender
Male
About me
-
My research interests
Neurobiology, Molecular Biology, Biochemistry, Stem Cells
Contact Information
1
Neutral
User Tools
Friends
Latest Visitors
Posts I've Made
In Topic: Stable protein expression in Vero cells
06 September 2012 - 11:57 AM
Is it not too much 1mg/ml of G418? Are you using the lower concentration to kill the controls? It sounds to me that you are using too much Neomycin and your cells are suffering...
In Topic: Why should I use glycine to a final concentration of 125mM ? why not more or les
06 September 2012 - 11:52 AM
That is why... not only the quenching. I never had problems with "exonuclease digestion".
In Topic: Fastest way to detect bacteria?
06 September 2012 - 11:40 AM
It depends on what you want to do. Do you want to check only for bacteria or you want to check if your bacteria have a plasmid of interest?
For the first the best it to growth a small culture (5ml) with SOC medium at 37°C and check with a spectrofotometer if the OD increase after 2-4 hours.
For the second, do as bob1 says. Pick the colony with a 10ul tip, inoculate a LB-agar plate to keep the bacteria and put the rest of the bacteria directly into the PCR mix. The first cycle of the PCR should be at 95°C for 5min to boil the bacteria and release all the protoplasm including the plasmid into the PCR mix. It is really fast (3h) and also very convenient if you have to pick more than 20 colonies to check if they have your plasmid instead to do minis like crazy.
For the first the best it to growth a small culture (5ml) with SOC medium at 37°C and check with a spectrofotometer if the OD increase after 2-4 hours.
For the second, do as bob1 says. Pick the colony with a 10ul tip, inoculate a LB-agar plate to keep the bacteria and put the rest of the bacteria directly into the PCR mix. The first cycle of the PCR should be at 95°C for 5min to boil the bacteria and release all the protoplasm including the plasmid into the PCR mix. It is really fast (3h) and also very convenient if you have to pick more than 20 colonies to check if they have your plasmid instead to do minis like crazy.
In Topic: positive NTC in real time reaction
06 September 2012 - 11:30 AM
No way, if you are having signal in a NTC and this signal is giving you the same melting temperature that your amplicon of interest your NTC is contaminated. The Melting temperature of non-specific amplifications or dimer primers should be different (usually one pick) and much lower , than the melting temperature of your product. Try to know if this signal is coming from dimers/non-specific amplification or contamination in this way. If you are having non-specific amplifications you should set up your reading step at a temperature 2°C higher that the melting temperature of the non-specific product, so you only will have signal from your specific amplicon.
In Topic: WANTED: Suggestions on how to decontaminate with formalin/formaldehyde
06 September 2012 - 11:21 AM
It sound familiar to my this problem...mmmm, The same in my previous lab. First, if it is possible you should clean your incubator with Deconex. Second, autoclave all the parts and also the incubator (I suppose that it has an autoclave program, hasn´t it?). Third, fill the incubator only with autoclaved ddH2O. It is not necessary to replace the water every week, only when levels are low. And forth and very important... check if your culture room is really clean. Spores will not be resistant to Deconex and autoclaving, or Virkon as you used, what means that you have a source of contamination from somewhere else... air-conditioned filters, dirty floor, not positive pressure in your room...
- BioForum
- → Viewing Profile: Posts: Akdor
- Privacy Policy
