Wednesday 21 September 2011
last post.......
Last week I finished my placement in the University, during the last week I attempted to do another in situ hybridisation in order to identify where my gene of interest was expressed. Although we managed to get staining I think we rinsed the sample very fast and the majority of the staining washed away making the slide really faint and hard to visualize under the microscope. Unfortunately it is a very tricky technique that you need to optimise for your own probe and mRNA of interest. The protocol states that the staining can be left between 10 minutes and 24 hours so, mmm hard to get it right. On my first attempt my friend and I allowed to stain for about 2 hours and the majority of our sample was really over stained and this time round we allowed the reaction to proceed for about an hour and 15 minutes and it was under stained!!!!! Shame I feel like we were so close to get it right but unfortunately my time in the lab has finished and now I got to prepare for my third year that starts next Monday!!!!
Last year I read a couple of comments before deciding to do a summer placement and now after having done it the only thing I can say is that if you are considering to do a summer placement and you are reading this.... go for it!!!! It is well worth it and I have learnt a lot, I feel more confident in the lab and more independent I feel like now I am ready for my third year project and possibly a PhD xxxx
Adriana
Monday 5 September 2011
last week....
Last week was very exciting because I did an in situ hybridization, something I had not done before in practicals. I did it following a protocol in a journal.
The purpose of the in situ is to localize the expression of the gene that I am interested in as well as getting some idea of the level of expression but since I had never done that before this first attempt was really just to get hold of the technique so we only did two different phenotypes and a control. We already knew where both genes were supposed to be expressed and our control just did not have a probe.
For the two genes one was slbo which is inlvoved in the signalling pathway of differentiation of border cells so I was expecting to see the antibody binding in border cells and that actually happened so im thrilled because of that. Our second gene was domeless and that one did not work very well.
We also experienced some problems with the control which was really disappointing !!!! Since my control only had hybridization buffer I was not expecting to see any antibody binding at all and I was expecting some clear ovaries but unfortunately I got some binding in the cytoplasm which makes us think that the antibody is not specific enough!!!!and is clearly binding to something else.
Before I arrived another student doing a summer placement tried to perform an in situ and he had a lot of background staining and non specific binding so my supervisor order some fresh antibody to rule that possibility out, unfortunately even with the fresh and preabsorbed antibody there was some binding.
I will have to talk to my supervisor about this issue but I might also try increasing the concetration of the probe and decreasing the staining time to try to get only very specific binding !!! I dont really know where to go from here so as soon as I know ill get to it.
I have also continued preparing some more slides everyday and I cna say my dissecting skills are really improving =) plus my slides look much better!!!!
xxxx
adriana
The purpose of the in situ is to localize the expression of the gene that I am interested in as well as getting some idea of the level of expression but since I had never done that before this first attempt was really just to get hold of the technique so we only did two different phenotypes and a control. We already knew where both genes were supposed to be expressed and our control just did not have a probe.
For the two genes one was slbo which is inlvoved in the signalling pathway of differentiation of border cells so I was expecting to see the antibody binding in border cells and that actually happened so im thrilled because of that. Our second gene was domeless and that one did not work very well.
We also experienced some problems with the control which was really disappointing !!!! Since my control only had hybridization buffer I was not expecting to see any antibody binding at all and I was expecting some clear ovaries but unfortunately I got some binding in the cytoplasm which makes us think that the antibody is not specific enough!!!!and is clearly binding to something else.
Before I arrived another student doing a summer placement tried to perform an in situ and he had a lot of background staining and non specific binding so my supervisor order some fresh antibody to rule that possibility out, unfortunately even with the fresh and preabsorbed antibody there was some binding.
I will have to talk to my supervisor about this issue but I might also try increasing the concetration of the probe and decreasing the staining time to try to get only very specific binding !!! I dont really know where to go from here so as soon as I know ill get to it.
I have also continued preparing some more slides everyday and I cna say my dissecting skills are really improving =) plus my slides look much better!!!!
xxxx
adriana
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