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There have been 2 items by vzp766 (Search limited from 30-May 19)


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#167578 DAPI stainged dots only in cells transfected with Fugene6

Posted by vzp766 on 06 May 2014 - 01:26 AM in Tissue and Cell Culture

Thank you!

So, we already cleaned everything in rodalon and ethanol, threw everything (media, PBS, cells etc.) away, decontaminated the incubators. Basically decontaminated everything, and started up from scratch. We also did a mycoplasm test twice and they all came back negative.

 

Together with dapi we also use Acetylated Tubulin antibody and we always see the cells look just fine, so I don't think they are apoptotic vesicles.

 

We did a new test where we mixed fugene6 and plasmid and dapi stained it. What we saw looked exactly like the dots we see. So probably it is due to bad fugene? The fugene-DNA complexes form, but they don't enter the cells. Wouldn't that make sense? 




#167556 DAPI stainged dots only in cells transfected with Fugene6

Posted by vzp766 on 05 May 2014 - 04:50 AM in Tissue and Cell Culture

Hi

We have a problem in our cell lab. When transfecting cells using fugene6, with different plasmids, we see blue dots everywhere (inside and outside the cells) when staining with DAPI.

 

Initially we thought it to be contamination due to the nice blue dots that appear all around the slide. However, we only see these dots when we transfect our cells and not in the untransfected cells.

 

We've already tested a few things:

- we see it in several cell lines using several different plasmids and we just opened a new bottle of fugene6.

- we don't see anything when adding only fugene or only plasmid (only when the two are combined).

- The cells are nice and viable and do not seem affected.

- When incubating either fugene, plasmid or fugene + plasmid in growth media, followed by incubation the weekend over, the media is still nice and clear.

- transfection efficiency is very low

- Results from other experiments where we transfect but don't do IFM (e.g. luciferase, qPCR etc) seem unaffected and look like they normally do.

 

Could it be vesicles with DNA due to poor transfection efficiency? Or some sort of infection which is triggered by fugene-plasmid complexes (sounds odd)? 

 

Please help!! 

Thx





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