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. 

Agarose, agarose and more agarose...

It has come to the end of my first week (I know I only did my introduction post yesterday, but y'know, time gets away from you when you're in the lab)-- and I've found myself to be quite the little busy soul.

On Wednesday, which ended up being my first day due to complications getting back from Spain, I was eased in to the lab environment with some bioinformatics work. Normally I'm not one for bioinformatics because it winds me up (I get stressed out in bioinformatic practicals like you wouldn't even believe); but I actually found this particular bioinformatics work pretty enjoyable. Probably because instead of simply database trawling, I was analysing the latest CHiP data that the lab had received back.

The data that I was given outlined points in which Zta (a DNA binding protein) is found to bind DNA (discovered experimentally by one of the PhD students in the lab). The specific binding regions were then mapped against the human genome to give two different possible data outcomes: unique peaks and repeat peaks.

It was my job to identify which of the peaks were unique-- which were then placed in a separate file which is going to be further analysed in due course (probably next week). The idea is for me to identify the unique peaks that have the least single nucleotide polymorphisms (SNP's) and that correspond most closely to the transcriptional domain-- ultimately for us to design a primer pair which can then be used to identify Zta binding domains.

Wednesday afternoon was my health and safety talk with the added addition of the lab tour. With the lab being split between three separate research groups, things are in very specific places so I've had to learn where to find all our equipment opposed to using anyone elses' by mistake. I think I know where most of the equipment is now (I've drawn myself a map) but only time will tell whether I will be able to remember how to read my map when it's not so fresh in my mind.

Anyway, I've been set up with my own little work space within the lab, although at the moment I tend to spend more time at Angelica's bench (the post-grad who is showing me the DNA ladders), just because I'm not quite ready to fly completely solo yet. Having my own work bench though sort of makes me feel all official and properly scientific.

Thursday was spent learning the ins and outs of Polymerase Chain Reactions (PCR) and Agarose Gel Electrophoresis. In total the experiment has 4 fragments of DNA that we're trying to analyse; all of which are overlapping (1-4)-- in our PCR we took fragment 1 and 2 and amplified them which, well, was a little bit of a fail because when we analysed it in the gel electrophoresis only fragment 2 presented and even then it wasn't particularly amplified.

I guess though, that's what happens with science. You're experimenting. You're not always going to get the results you want (although, for my first PCR I would've liked a little encouragement that I was doing something right!)

In the gel electrophoresis we were testing fragment 3 and 4 on an extremely large scale-- so we ended up making giant agarose gels for them to run on. I didn't even know they existed in this sort of size! The idea of this experiment was to determine that the two fragments were of different sizes and no cross-contamination had occurred as well as providing us with an isolated DNA sample that we could then purify.

As it happens, this worked extremely well. We ended up running 3 separate gels which all showed the desired results which just helped to solidfy our conclusions (one gel with just sample 3, one with just sample 4 and one with sample 3 and 4 side-by-side):

  Sample 3 and 4 side-by-side gel 

(sorry about the quality, it's off my phone opposed to scanned)

After running the gel, I then had a brief experience of feeling a little like a member of the CSI team. We had to take the sample up to the UV room to cut the agarose regions that contained DNA out-- which obviously means full protective gear including the face-shields (hence the CSI comparison, it does make sense). Nervous doesn't even begin to cover what I was feeling faced with UV-- I guess that because it gets drummed into your skull how dangerous it is, when faced with it I was more than a little bit wary. Of course, there is no need to be really if you're careful, so separating the samples from the agarose came off without a hitch.

Today I was taught a method of gel extraction which would allow us to purify the DNA and separate it from the agarose gel. The procedure was pretty easy to get your head around (everything came in a handy little separation kit, which makes life that little bit easier). It bascially consists of dissolving the agarose gel in a buffer and then centrifugating the resulting mixture to separate the two elements.

We then ran the resultant elements of sample 3 and 4 against already purified samples of 3 and 4 (done in a previous experiment prior to me arriving) on a small agarose gel and got absolutely perfect results:

Ladder, Purified 3, New 3, Purified 4 and New 4

This means that on Monday we can set up Western Blot for sample 3 and 4-- which although it seems like it's going to take a long time, looks like it should give exactly the results we're aiming for.

Theoretically of course.