moerae

I wasn't sure whether I should have this in my previous post as this is a different issue. I still haven't been able to fix the QC issues and as this is an optional control in the kit we're seeing if getting bad QC score still allows us to detect deletion/duplication events. So I've tried it on control samples that have one gene that contains a duplication/deletion, and did these in duplicates. The QCs fail (as expected now), but the array is detecting a mutation event in the gene of interest (GOI), but for some of the samples instead of detecting a dup, the array would call it a del. To top it off.... a lot of GOI for my project is being called to have dels/dups and this is just not believeable to me, we're expecting one or two for each sample. These same "calls" are appearing in many of my samples. Contamination? I've been careful with how I perform the reactions so I'm a bit boggled as to wear the contamination could come from.

If someone has any ideas... I'm all ears!

Hi all,

I'm new to the whole microarray scene and I'm having real issues with getting my arrays to pass QC. I'm using the Nimblegen CGH DNA arrays and they have an optional Loading and hybridisation control which is causing me grief. This is basically the proxy measure for either a duplication or deletion event seen in the samples, and also showing how well the hybridisation and labelling went. I've checked the DNA quality of my samples before hand and they're all fine and not degraded. And I'm being careful with keeping the labelled samples covered in foil and away from light, which the rep told me could affect the QC. However, my samples are still not passing QC after I've tried again and being careful with light exposure and etc.... Does anyone have any ideas as to what else I can do or am doing wrong that might help?

Hi,

I'm trying to over-express a protein in zebrafish and I have them in a vector that doesn't have a polyA signal. I will eventually clone it into a vector that has a signal, but I want to just see whether the expression of the protein is correct first. I was going to do an RNA run off and inject into the zebrafish. Will the lack of polyA signal affect the expression of protein?

Hi,

I'm trying to embed live zebrafish embryos (48 hpf) without using Tricaine/MS-222 (as it affects my results). I've tried embedding them in agarose, but it deprives the embryos of oxygen which is an improtant factor that affects my recordings. Does anyone know of any other method of immobolisation that would not deprive them of oxygen?

Hi all,

I'm trying to see whether an antibody targeting a rat protein would cross-react with the zebrafish equivalent and save me a lot of hassle. I've done the Western with different dilutions of the primary antibody (1:100, 1:250, 1:500 -- the recommended concentration is 1:100; used 1:20,000 secondary antibody)) and have run a rat tissue control as well as a zebrafish sample and there's a lot of non-specific bands even for the rat tissue sample (and they are bright!), which doesn't inspire much confidence. Is there any way to decrease the amount of non-specific bands showing up? I blocked with 5% skim-milk powder for 4 hours and probed with primary antibodies overnight (should I cut down on that?). Probed with secondary for 1 hour and developed the blot.

Another thing is that the bands are quite fuzzy. I have no idea why that is. Is there any way to fix that too? I make my own 12% SDS-PAGE gels with 4% stacking, and run them using Tris-glycine buffer at 200V. My transfer buffer contains Tris, glycine, methanol and 0.03% SDS, and I transfer for 1 hour at 100V onto PVDF membrane.