Andrés_Inuznza

Hello there. I've been having a really hard time with my cultures of human immortalized podocytes. Everytime I put them under differentiation conditions, they die because of contamination. For more context, this culture is expanded in DMEM F12 10% FBS at 33°C until confluence. Then, for differentiation, it's subcultered into some plastic previously coated with laminin/fibronectin and maintained with RPMI 1% FBS 1X ITS at 37°C, with media changes every other day. When the cultures are expanding they're fine, but when I start the differentiation they die before a week, that's to say, about the second media change. Cells look detached, media looks cloudy and slightly basic, and I've seen small dark dots, so I'm guessing it's bacterial contamination. No other culture at 37°C gets contaminated, we've prepared new media, new PBS, I've thawed several vials frozen at different times and everytime I get the same results. So, do you think it's bacterial contamination? Is it possible that the source of contamination is the laminin/fibronectin solution or the ITS? Obviously the problem starts when that is used (I have some other podocyte cultures that are not contaminated when expanding, even for weeks) and those are the only reagents that haven't been changed, could bacteria resist there? Than you in advance

Hello everyone. I started to work in a lab, where the focuses are molecular ecology and evolutive genomic. One of the projects is the genomic characterization of different samples of Pyura Chilensis, because we think that there are different ecotypes among the samples we're studying. I'm an undergraduate student at Universidad Austral de Chile, currently studying Biochemistry, and my aim into this laboratory and project is to help the team with their biggest problem: obtaining high quality, pure and good concentrations of gDNA from the Pyura samples. When we extract the DNA, the result is completely degradated DNA (we can tell it by doing the EPs). We've allways used GeneJET Genomic DNA purification Kit (by ThermoFisher), and when we use it on other samples (I mean other species) it works beautifully, so the kit isn't the problem. We've always used tissue storaged, but the problem always appears even if the samples are one day old (and storaged) or even years old (We use Ethanol 95% for storing, and we wash the samples with Ultra Pure water before starting the procedure with the kit), but the storing method seems to be good for extracting gDNA in other species. Then, my guess is that the problem is the Pyura itself (lol). So I have to figure out if either the extraction or the storage isn't appropiate for Pyura. I read that this Pyura thing has a lot of cupper within it, then we're going to use a new buffer for storing it (Salt Saturated DMSO, containing EDTA), and I'd like to perform another methods for the DNA purification (something more classic, like phenol/chloroform or something). Finally , after the tedious contextualization, I would like to ask if any of you have ever worked with this organism (and if you have, what did you do and how did you managed it. I think that most of the time there's someone more experienced, and perheps someone already went through this) and what are your recommendations for solving my problem. If you know any technique for obtaining the DNA the way I described you (we need to sequence it), ways of storing (for example, would it be better if we extract the DNA and we store it instead of the tissues? Once we achieve the way of obtaining good DNA, of course; or if you think that the new buffer will work; or your recommendations when we do PCR because of the EDTA), or any advice, anything will help me and I'll be profoundly grateful (if I can achieve my aim, maybe my professor puts it into an article and my name appears on it, such a thing for an undergraduate hahaha). Thanks in forehand for reading and helping (and excuse me for my bad english, it's not my mother language). Greetings. PS: I added an image corresponding to the result of an electrophoresis with the product of the extraction. 1st lane is the standard (1kb), then next 9 lanes aren't my samples, and then those 3 lanes with long blurred thing instead of bands are the DNA I obtained. Last 3 lanes weren't loaded