I didn't end up updating last week like I'd promised myself I would. Things got extremely busy around here what with the lab, my normal job and attempting to pack, I barely got time to write my shopping lists nevermind a whole blog post. So apologies about that!
Anyway, last week was all about running what should have been the defining western blot. Note that I said should, because it didn't quite go to plan, but I'll explain that later.
On Monday we started off by doing some DNA 'pull downs' using a DNA affinity protocol. For this we added our purified DNA (samples 3 and 4) to streptavidin beads. The theory is that the DNA should bind to the beads whilst the rest of the supernatant can be removed from the sample.
When we tested our supernatant in a gel electrophoresis we got a partially successful experiment. Sample 3 showed significantly less banding after the 'bead washes' than prior which meant that the fragment had bound to the beads. Sample 4 showed slightly less banding after the bead washes, but there was still quite a large intensity to be found in the result meaning that it did not bind as well as sample 3.
Although this result was not the best in the world, we decided to continue with the western blot analysis. It may seem a little backwards to continue on-- but sample 3 is one of our OriLyt fragments whilst sample 4 is actually a control DNA fragment, which means that as long as sample 3 showed a correct result the rest was disregarded. (Ideally you wouldn't, but time constraints of the Postgrad I was following pushed us to continue).
On Tuesday once I'd found out I officially passed my second year, we added our nuclear extract to the streptavidin beads that had our DNA on that we had prepared the previous day. Due to the consistency of our nuclear extract we had to spin the extract prior to its' addition and we are now reasoning that this could be what caused the rest of the experiment to go a little awry. Anyway, theoretically what should've happened is that some of the nuclear extract (with the elements that the EBV OriLyt binds to) should have remained attached to the beads in sample 3, whilst in sample 4 nothing should have bound.
A whole lot of antibody amplification, incubating and blocking (with milk, winning the most unexpected thing in the lab ever) we end up at Wednesday afternoon, when we went to develop the blot. First thing I have to say about developing a western blot is that it's so difficult to do in the dark when you have no clue as to:
a) How the room is laid out
b) What the procedure actually involves
Being taught in the dark is a bit of a surreal experience, just let me say that much!
Once our films had developed, the result was extremely disappointing. We had no bands at all which for sample 4 would be ideal, but the lack of anything in sample 3's lanes put a bit of a dampner on proceedings.
The lack of bands can be explained in one of two ways. Either in spinning our nuclear extract we degraded some of the binding or that the concentrations of the nuclear extract binding is far too low for us to observe on the western blot. It's a bit of a mystery unfortunately.
On Thursday and Friday I returned to some of the bioinformatics work I've been given to do intermittently over the weeks. After identifying the unique peaks I have been given a GO LIST which contains a list of genes that are seen to be associated with the peaks. Now I've been tasked to identify the positions of these peaks with relevance to the genes and to also identify whether there are DNase clusters associated and whether there is any exposed chromatin in the region. We're hoping in by sorting all this data out we'll get a clearer picture of which genes we should start experimenting on in detail.
This week I am helping out on a different project whilst my supervisor is away. So I'm now having a go at site directed mutagenesis and transformation of E. Coli. I will go in to more detail about that in another post because I don't want this to become a too threatening wall of text.
Until next time...
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