moblog uk

Spidermonkey's Sporadic Lab Stuff

by Spidermonkey

user profile | dashboard | Spidermonkey maps

Hello World from the lab!

Part spider - part lab monkey, will do science for cash, strawberries, or for a place to be that's out of the rain (but preferably for cash).

The rest of what I get up to goes into the Tunnel of Goats :)

Urgent Science Stuff:

Please help reform English libel law
Current UK libel laws are very bad for science and free speech.


Cool science stuff:

RCSB Protein data bank: Molecule of the Month in alphabetical order

The PCR Song It's the little things that keep you going...

NCBI's Entrez Gene My favourite starting point for finding out what is known about any gene of interest.

NCBI's PubMed Where to go to find pretty much all research published in the last 30 years or so (may go back a lot further now). All newly published research is quickly added to the site.

The Genetic Code - table of the DNA/RNA triplet codes for amino acids. This is how DNA codes for protein.

Another PCR song Disco frenzy :)

Recent visitors

Friends

More...

rss rss feed

Holly and Ivy?

(viewed 576 times)
A little late perhaps but these did look so pretty as I was cloning this morning (well, trying to get a particularly obstinate bit of DNA to amplify so that I can then clone it into bugs) :)
4th Jan 2010, 18:44   comments (3)

I jumped for joy

(viewed 637 times)
...because my competent cells were not all contaminated with a rogue plasmid after all, so I don't have to make them all again. Turns out the antibiotic had gone off, making a new batch worked perfectly. Big relief :)
1st Jan 2010, 22:36   | tags:comments (5)

New lab partners

(viewed 493 times)
From now on I will be ably assisted by the three Doctor Ducks. I am expecting great things.
28th Dec 2009, 02:27   comments (2)

More cytoplasmic?

(viewed 458 times)
I think so, in some cells at least... Hard to tell in this cell type, as the cytoplasm gets all squashed up when they grow close together. Next trying a couple more cell lines to see if the picture is any clearer in any of them. These are 293T/17 cells.
28th Dec 2009, 02:22   comments (0)

Something is different

(viewed 433 times)
This was the first test of the thing I have been so desperately trying to clone over the last few weeks (feels like forever!).
I transfected the plasmid into HuH7 (liver) cell line into some of the wells on a 12 well plate, and control plasmids into other wells. The top 2 images are from 2 different wells that got my plasmid. I was hoping that they would look like the cells in image 3 (where most of the green is in the cytoplasm, the dark ovals being the cell nuclei), so that's a shame, however they do look different to the GFP only control well (image 4), where the green is kind of diffuse and all over the cells) so something at least has changed. A lot of the cells with my plasmid have what look like clumps of very bright green, which is most likely due to the protein getting stuck somewhere in the cellular machinery where proteins are assembled.

Trying this out in a different cell line to see whether or not a different cell type will be better at processing the protein. By better, I mean, give me a phenotype more like that in the 3rd shot. If that works, then I can try treating the cells with chemical compounds known to bind to this protein and see if the fluorescence moves into the nucleus. And if that works then I will have a model I can use to test other compounds to see if they, too, will be bound by my protein and are therefore likely to have a biological effect downstream of this pathway. In other words, then I can actually do an experiment! :)
18th Dec 2009, 18:26   comments (1)

Name that cell: round 2

(viewed 536 times)
These cells have been transfected with a standard green fluorescent protein (GFP) vector. The top image shows what you see when you look through the green filter, the middle image is the bright field image (no fluorescence, shows you where all the cells are), and the 3rd one is a merge of the 2 previous images. Merging the GFP and bright field allows you to see what proportion of cells in the flask actually took up the GFP plasmid.

These are a different cell type to the previous set (293T/17). These have a polygon appearance, having mostly 4-6 straight edges, whereas the 293T/17 cells have more spindly processes growing out from the main body of the cells.
18th Dec 2009, 04:41   comments (1)

Titrating transfection reagents

(viewed 479 times)
These pretty green cells have been transfected with plasmids for Copepod green fluorescent protein (a slightly different green to that of standard GFP from jellyfish, and brighter too), and for the packaging proteins necessary to make lentivirus. The virus made here will infect other cells and make them glow green. This is just a test to find out the best mix of transfection reagents to use. The main aim after this is to make virus that will make infected cells express my favourite protein tagged with GFP. This is going to be for use with primary cells (eg from liver tissue donated for research use). Such cells are very difficult to transfect (by adding plasmids) so viruses are necessary to get your gene in there.
It means working at a higher level of biosafety, but I work at that level with all my tissue culture anyway for piece of mind. Keeps them uncontaminated by me and vice versa :)

For anyone who can recognise the cell type, I'll be awarding points. And what do points mean? :)
14th Dec 2009, 00:49   comments (12)

The test

(viewed 438 times)
Extracted plasmid DNA from positive clones (identified yesterday). Now added them to some liver cells to see if they a) make the cells go green and b) make the cells go green roughly in the right place (in the cytoplasm rather than in the nucleus or all over). Tomorrow will tell. I daren't get my hopes up, and yet...
10th Dec 2009, 02:16   comments (0)