Sunday 21 August 2011

Flying solo in the lab obviously means you're going to destroy something...

As predicted, this week I returned to my analysis of the origin of lytic replication in EBV-- although this time I was doing everything completely by myself. As exciting as it is to be told that you're now 'flying solo', it's also quite daunting. After all, what happens if I get confused? What if I don't get results? What if I blow something up? 

Okay, so blowing something up is extremely unlikely in this instance. But who knows, maybe I was going to be the one to short circuit something and burn down the entire lab. (I have a very over-active imagination, it has to be said).

Nothing like this did happen, of course. The only thing that I have managed to prove is that no matter how careful I am, I seem to be unable to get a successful PCR reaction. I've gone head-to-head with it now four times and got: 1) contamination like you wouldn't believe, 2) a positive PCR for one of the four samples, 3) no amplification at all, 4) a negative result on my gel. Round five will not be pretty unless I get some sort of positive result, it's starting to aggravate me.

PCR is not the only thing that I've been doing this week (otherwise, well, I may have lost it completely and be a gibbering mess of a failed scientist by now), I've been busy with my first OriLyt pull-down!

On Monday I was given the task of taking apart the two different assays that have been used in the past to do an OriLyt pull-down and decide which of the techniques give a better result and would be better to use. The first assay was the one suggested by the company that provides the Streptavidin beads (Invitrogen) and the second assay was designed by the masters' student that has just finished her project.

Although the basic outline of the procedures are the same-- the concentrations, timings and some procedural steps are different (where the text is normal, the procedures match, where there is bold this determines what Invitrogen suggest and italics indicates the masters' assay)

  1. 20ul of beads to magnet and removal of supernatant.
  2. Addition of 200ul of bead-bind wash buffer. Remove from magnet, wash.
  3. Repeat step 2 three times.
  4. Addition of DNA to beads/ Resuspension of beads in bead-bind wash buffer to 5ug/ul concentration (40ul)
  5. Addition of equal volume of biotinlyated DNA (diluted in H2O) to buffer.
  6. Wheel for 3 hours/ Wheel for 15 minutes to 1 hour.
  7. Centrifuge/ Apply to magnet and remove supernatant to rewash (once)
  8. Resuspension to desired concentration
  9. Gel-electrophoresis.

Once the gel had run and developed, I found that although both assays significantly reduced the DNA concentration from the original sample (indicating that there had been successful biotin-DNA binding to the Streptavidin coated beads).

 Sample 3 original (lane 2) with Masters' Assay output (lane 3&4)
Sample 3 original (lane 5) with Invitrogen Assay output (lane 6&7)
Sample 4 (lane 8) to allow concentration estimation

Looking at this, I decided that using the suggested method by Invitrogen would be the way forward as although we had the same approximate ratio of B-DNA binding, the procedure only required a maximum of 1 hour incubation on a wheel which meant that a full bead-DNA-nuclear extract preparation could be done within one day.

The following day I repeated the chosen assay with sample 4 just to ensure that it wasn't a fluke. Although the gel didn't run properly (soon to be discovered that there was contamination with the TBE-- overnight seemingly) there was still a positive result:

Sample 4 original (lane 3)
Sample 4 with bead addition (lane 4)

After this I was tasked to make up the buffers that are required for the second portion of the assay for the completion of DNA affinity protocol. This involved a lot of maths (which I quite frankly fail at doing every time) and weighing as well as some precise pH work (as the buffers have to be at pH 7.9). I was also required to work out the concentration of Did-C using spectrophotometry which was a bit of an eye opener as to how sometimes translating 'units' into usable concentration is more awkward than you'd think.

On Wednesday I spent the day working with the nuclear extracts to complete the assay. First I was tasked with spectrophotometry to determine the OD280 of the samples-- proportional to the amount of DNA/Protein present in the extracts. This was done so I could add a proportional amount of each different nuclear extract to the beads from sample 3 and sample 4 (I had three different nuclear extracts from 3 different cell-lines). 

After preparing the nuclear extract appropriately, they were then centrifuged and 3ul was removed to run on a gel at a later date. 30ul of each nuclear extract was then added to their appropriate beads (resulting with: NE 1 with Sample 3 and Sample 4; NE 2 with Sample 3 and Sample 4; NE 3 with Sample 3 and Sample 4-- amassing to 6 separate samples). These were then rotated together for 45 minutes before the supernatant was removed (which has been frozen to run on the same later date gel-- allowing us to pick apart the whole pull-down procedure if needed). The samples were then washed with a buffer before being added to Protein Sample Buffer and boiled.

This boiling procedure allowed my microcentrifuge tubes to gain coloured shoes.
These are geekily adorable. 

At this point all the samples have been frozen ready to be run on SDS-Page and a Western Blot early next week to show whether we had a successful pull-down or not. Right now I am keeping everything crossed that I have a positive result so I can go on and analyse the remaining 2 OriLyt fragments (Sample 1 and 2). 

The rest of the week was spent on aforementioned PCR-- but we all know how that went, so it's not particularly worth going in to any details when obviously said details aren't working the way they theoretically should

Sunday 14 August 2011

Mutants, like X-Men, except in E.Coli.

As I mentioned in my last post, I've been temporarily helping out on a different project whilst my supervisor has been away. This time I was helping out with site-directed mutagenesis, transformation of E. Coli and preparing bacterial mutants for sequencing.

This was working alongside one of the PhD students in the lab, giving me a bit of an experience of what a PhD involves. Looking at it now, I've got to say that it's something that I would be highly interested in doing at some point-- but it is a decision that really shouldn't be taken lightly because it looks like a lot of hard work.

The first day of assisting on this project, I found myself learning the ins and outs of tissue/cell culture. The whole point of the tissue culture is to ensure that at all times there is an established cell line that can be used in the experiments. So I helped out examining the current cultures to see how well they were growing (that's a fancier way of saying that I was counting cells) and sterilizing all the equipment that was being used (as it's all highly open to contamination and when you're trying to grow a cell line this is exactly what you don't want).

After this, I was introduced to the procedures for site-directed mutagenesis-- using PCR. Nick (the PhD student) had designed and had his mutated primers already made, so it simply was just a case of adding the required components and setting the PCR to cycle through the conditions required to cause high numbers of the mutant plasmids to be replicated.

Once the PCR had completely cycled (4 hours later) we then removed the products and discarded the wild type plasmids using a digestion (in which the unmethylated wild type, normal plasmids were removed leaving only the methylated, mutant plasmids behind). We then transformed highly competent E. Coli cells with the plasmids and allowed the cells to incubate overnight.

The following day I was taught how to produce selective agar plates. Normally I would be completely up for doing any practical work-- but unfortunately when you produce agar plates you have to be fully suited and booted to stop any cross-contamination to the plates and you also have to have a bunsen burner going at full whack-- and it was probably one of the warmest days of the summer. Literally so sweltering, I don't think I've been that warm since my tent in Spain.

Anyway, to make the plates a selective medium, we added antibiotics to them which would ensure that only the E.Coli that had taken up our mutants would grow. If the colonies did grow then we'd know that we did, indeed, have a successful mutagenesis... Which we did! (For me that was a little bit of a novelty as I've not exactly had the best results thus far).

Over the next few days we repeated the above procedure several more times for different mutant lines (CIITA Forward, CIITA Reverse, CIITA negative, Zta Forward, Zta Reverse and Zta Negative) which are helping to amass a picture of the protein components of EBV.

Once all of the different mutant cell lines were made, I then took the remaining bacterial cultures and set about extracting the DNA elements from the cells via mini-preps. First for this the cells had to be isolated from their growth medium through centrifugation (the first time I'd ever used a large centrifuge opposed to a table-top one)-- which gave a pellet. The pellet was removed and then resuspended in 2 separate buffers (with Lysate Blu in which gave a colour change to confirm the procedure was being carried out correctly). These solutions were then placed into a table-top centrifuge and spun-- which caused the cellular elements to become debris leaving us with only the soluble components (proteins/RNA/DNA).

After removing the soluble elements I then carried out a separation procedure extremely similar to the procedure I'd used to separate the DNA from agarose a few weeks ago. Having done it before, by the time I'd finished the extraction this time around I felt like a tiny bit of a professional because obviously I could do it at a relatively steady speed without asking for any clarification.

Once the DNA had been purified, I then made a 1 in 10 mastermix of the DNA (diluting it with double-distilled H2O which, I didn't even know existed) which was then analysed via spectrophotometry. This gave us the overall concentration of DNA within the mastermix-- which I then used to calculate making a 50-100ug/ul solution. These resultant solutions have now been sent off for sequencing and they'll hopefully be back at some point this week.

Oh, also at some point over the last week this sign has appeared on our lab wall. It instantly became clear why a career in scientific research is for me:


Anyway, it's hard to believe I'm already halfway through my studentship. It's going extremely fast and before I know it I'm going to be submitting my report (slightly nerve-wracking in itself). Next week I believe I will be returning to my analysis of the OriLyt fragments as my supervisor is back, so I will obviously update to let everyone know what results I'm getting on that front.

Tuesday 2 August 2011

The blots that don't blot...

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...