9 replies to this topic

[SunsetSatine]

I've been trying to research to see if the primer 27F and Anti-Gamma primer are the same thing, but when I try to google Anti-Gamma primer, I don't get anything relevant to my search.

I have an isolate that did not amplify with 8F and 1492R, so I thought I would try 27F and 1492R, but we don't have any primers labeled 27F in our primer box. We do have one labeled "Anti-Gamma" and I wondered if that was the same thing.

Thanks!

[bob1]

Your best bet would be to get the primer sequence and blast it.

[SunsetSatine]

I was hoping to find the primer sequence as well, but finding any information about the primer has been difficult.

[El Crazy Xabi]

anti-gamma? sure it is a primer? if you google

"anti gamma primer"

only gives this forum as result. It sounds pretty weird for me... any anti-thing use to be a antibody but a primer.... it's actually the first time I read it

Both 8F and 27F are universal primers so probably your lab only uses 8F, I guess. Are you sure your isolate is a bacteria? Positive controls are working?

[SunsetSatine]

Hi,

Yup, my positive controls are working. I did that extraction with 3 other different isolates, and they all worked with strong bands for the PCR. The nano drop has the isolate concentration at 15 ng/uL. I wonder if I have some kind of inhibition problem and if dilutions will help.

[El Crazy Xabi]

Try a 1/10 dilution of the DNA extract if there is a inhibitor problem, dilution can help. Did you see under the microscope if it is really prokaryotic? Some yeast colonies look completely like bacteria.

If it is indeed prokaryotic and dilution doesn't help you can try cleaning the DNA, but it is a little bit more work.

[SunsetSatine]

Under the microscope, I did some rods. I didn't even think about yeast! *headdesk* The sample came from somewhere else, so maybe it is contaminated. How does yeast usually look? Does it by any chance change color after a couple days?

[bob1]

Yeast will look like little ovals or spheres, typically with smaller versions coming off the sides (budding), they are quite a bit bigger than bacteria.

[SunsetSatine]

Hmmmm, okay. I'll go back and look underneath the microscope again. How do they tend to appear on agar plates?

[El Crazy Xabi]

At high magnification you can distinguish intracellular organels, at least the nucleus. They are eukaryotic so, usually much bigger.
In agar they can resemble a smooth rounded bacteria-like colony or something more "fungish" like Actinobacteria
https://www.google.c...SNM6iigejm4DgDw

Which medium are you using?

Also, is it slimy? Slimy bugs can give problems on PCR due to the huge ammount of polysaccharides/EPS produced...