Spot tests and pipette tips

Today I came in early to start my western blot from my gel that I ran yesterday.  I arrived to find I was the first to get in.  Think I might be a little too enthusiastic about this whole placement!  

Having located the nitrocellulose membrane and blot paper, which I was pretty chuffed to find, I set my blot to run, and panicked because my protocol and the top of the blot machine told me I needed different voltages.  Fortunately my memory served me correctly and it seems to have transferred well, from staining up the gel.  

I practically skipped to the incubator where my spot tests were, hoping that they had grown and worked and looked good.  For those who don't know what a spot test is: basically you pour some agar and let it set, then mix more agar (but more wobbly agar) and growth media (essentially like a very weak liquid version of marmite (well it contains yeast extract in any case)) with the cells you want to test, and pour that on top of your agar.  You then let the layer set and drop your bacteria killing compound on in different concentrations- mine were diluted by ten each time.  Once you've left it in a warm place overnight, the cells should have grown, but hopefully not where you've put your antibiotic, where there should be a clear spot.  

A revelation for me this week was finding out that antibiotic doesn't necessarily mean the type of thing you get at the doctors, but more anything that is anti something biological, so in  this case, my protein was the antibiotic.  

Anyway, my spot tests were pretty cool.  They seem to show that with my protein in a high enough concentration it can kill mutants we weren't expecting it to.  I tested the whole molecule, then 2/3 of it and then the last third on its own, so 12 spots on each agar plate.  I will try and put a picture up of the plates at some point.  I'm really pleased with this, and am testing the other two mutants, which theoretically should be partially killed, but not fully, tomorrow.  

It's nice to have something visual to look at, it shows what you know is happening in a way that you can immediately understand.  Not that I don't love gels of course, I don't think protein analysis could happen without gels.  Also they go really strange and solid if you let them dry by themselves!

I mentioned pipette tips.  We get through them at a rate of knots, as you have you use a clean one every time you pipette a different solution.  Spent this afternoon, whilst waiting for my agar to set, putting fresh ones in coffee jars (every lab seems to use coffee jars, I can't fathom getting through that much caffeine!) ready to be autoclaved.  Might have gone a bit overboard.  I reckon we've got enough for about the next month now!

 

Tomorrow I will finish my blot and spot tests, and move house, so it's going to be busy!  Not sure what my blot is going to show, but hopefully it might flag up some differences in concentration of the protein that I'm looking at.

New week, new conditions.

So, it's already Tuesday of my second week on my summer placement.  The days have gone so quickly!  Having had an exhausting but amazing weekend swing dancing, I was ready to see if my cells wanted to cooperate this week.

As you may remember, on Friday, I developed my blot, to find........  absolutely nothing at all.  The marker had just about transferred, but that was it, despite having looked at the gel, the bands I wanted had transferred onto the nitrocellulose sheet.

So, this week, like the weather here in Newcastle, I'm changing the conditions.  I'm adding salt, to make sure my reaction isn't electrostatic, as it looked like it could be that from staining the SDS gel.  I'm concentrating the samples more, and I'm using 10 well gels instead of 15, as you can get more sample in each well, which should give a clearer band.  

And today.......  I HAVE RESULTS!!!  This was a very pleasing finish to my longest day on my placement yet.  The bands are a lot clearer than the last, (though I think I made my gel a bit strangely, as the bands are a little wavy (I blame the acrylamide)) and the cells/proteins are doing more of what we thought they might do.  Overall a good day.

Even better, I'm looking forward to the results of my spot tests tomorrow, which I hunched myself over this afternoon, in order to make the perfect circle of liquid on my lovely smooth agar.  I've also found a use for my very useless skill of being able to write in mirror handwriting:  labelling the bottom of plates, so that I can see what it is when the plates are the right way round.

I also seem to have (finally after 4 years) mastered using moles.  For some reason I've always freaked out the minute I look at them.  Today's calculations were a success, without major panic.  Must find my own calculator though, Casio calculators just aren't my thing, thanks to a very determined statistics teacher that insisted on us using Sharps.

Will update on how the spot test and tomorrow's blot go soon.  Could be a while as I'm moving house this week, but I will try and post tomorrow.  

Week one. Proteins, mutants and teenagers...

So this week has been the start of my placement.  As I already know my way round the lab (I'm am so grateful for this, it usually takes weeks) I have been able to get straight on with doing things!  I've been growing up cells, mainly E. coli mutants, and adding proteins to them.  My agar even worked.  I have a history of agar hating me.  I can make it fine, and if it has come straight from autoclave it's great, but that rarely happens, so the 'melt in the microwave' technique is the best option, and either I don't heat it enough and it's lumpy, or I heat it too much, then panic that I'll denature the antibiotic and then leave it to get too cold, and it's still lumpy.  However, thanks to being made to practice earlier this year, which I'm very glad about, this week's agar worked really well.

This brings me on to word of the week, which is Kanamycin.  I know it's only an antibiotic, but it sounds great!!!

Panicked over my SDS page gel yesterday, which unusually decided it was going to take twice as long as usual to set.  The gels ran really well today (well after I was reminded to boil the samples anyway).  For anyone who doesn't know what the gel is, it allows you to separate proteins of different molecular weights using an electrical current.  The smallest proteins travel faster so end up at the bottom.  You can stain the gel and compare the bands that appear with a marker that has known molecular masses in it.  This helps you to identify if the protein you're looking for is there or not.

Today I was particularly happy as I started my first western blot ever.  This is where after running proteins on an SDS page gel, you then use electricity to drag the proteins onto a sheet, to which you can add antibodies to probe for the protein you want.  I was surprised to find that the process involves milk powder!

Feeling a bit heady due to pouring over my gel to see if we could find the protein, and breathing in a bit too many gel destain vapours!

I've also done loads of reading, and feel a bit more clued up on the subject of my project, there are so many things to know about just one protein, you'd be surprised.

I included teenagers in the title, as today we had a school visit, which the whole lab helped out in.  It was a microbiology based set of practicals.  I took great delight in telling them they were looking at the plague, anthrax, MRSA, TB, and various other lovely diseases.  Not so sure they appreciated it so much...  Also got the opportunity to judge the 'design a microorganism' competition along with Alex, a masters student in the lab.  There were some amazing ones, including ones that give you a six pac; clean your intestines; turn your hair into cheese; made you happy;  and made your eyeballs explode, but most of all, made you die a slow and painful death.  We ended up choosing the two that didn't actually kill you.  Had a really fun day and managed to do quite a lot of lab work in between.

Looking forward to finishing my western blot tomorrow.  I will find out if my first set of experiments have worked.  So far the only thing I really know is that my basic lab skills haven't been terrible so far!  I'll write another post, hopefully early next week about how it's all  going, and perhaps make it a little shorter!

First Post!

So, not much to say, other than that my studentship starts on Monday.  Really excited to be back in the lab, especially for this studentship, but also because I know that everyone there is lovely!!!  Promised I'd take in home-made cookies on Monday, so must not forget... that and my sharpies and calculator!