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Spidermonkey's Sporadic Lab Stuff

by Spidermonkey

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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 :)

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Yes! (maybe...)

(viewed 359 times)
As a last resort I went back and PCR checked all the tiny, tiny colonies that came up after about 2 weeks of incubation, just in case any of them had my piece of DNA I've been trying to get them to take up. I used primers (short pieces of single stranded DNA) that should recognise the beginning and end of the sequence and used to PCR to amplify any sequence that was there. And this is what I got. The bands are about the right size (same size as the positive control anyway (bottom right, last well). Streaked out the colonies onto a fresh plate so am hoping that they will grow some more over night. If that works I can start to bulk them up and then extract enough DNA to do an actual experiment on some real mammalian cells! An actual experiment! Feels like a long time since I've done one of them, rather than just trying to get my toolbox in order :)
Still keeping everything crossed.
27th Nov 2009, 22:45   | tags:comments (2)

These are still not the clones you are looking for

(viewed 367 times)
But they do help me figure out where the problem(s) are. The new ligase is a lot lot better than the old stuff, so at least I know that bit should work. Unfortunately it still isn't working with the pieces I want to stick back together. It is possible there is a mistake somewhere in the sequences used to make the bits of DNA in the first place, or for whatever reason this bit of DNA adopts some structural conformation which makes it stick to itself rather than to where I would like to stick it...
Leaving it here for today.
At least my luciferase assay made a change and has me thinking about something else I don't understand.
Now to go and help in the kitchen!
26th Nov 2009, 16:50   comments (2)

Third time lucky?

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Please, please work.
25th Nov 2009, 23:15   comments (2)

A bit more of this and that.

(viewed 636 times)
This being docking and that being gels.
I think I'm getting the hang of displaying the protein so you can see how the ligands fit inside, but I'm not happy with it yet. I think I'm missing something...

Got nice bright bands to cut out on the gel though, and only 2 of them so was relatively quick and painless to process. The gel is made with a tiny amount of ethidium bromide in it which slots nicely into the DNA molecules and glows fluorescent orange when you shine UV light through the gel. It's a bit carcinogenic but easily the best way to see where your DNA is on a gel, you just have to handle with care (like everything else in the lab really). The blue dyes are so you can keep an eye on how far your sample has run when it is still in the electrophoresis tank.
This was taken from behind a perspex screen to keep me safe from burns and eye damage.

Got new ligase today so have set up more ligations overnight. Will find out if these have worked Thursday morning.
25th Nov 2009, 03:06   | tags:,,,comments (6)

Today i am mostly modelling

(viewed 400 times)
It makes a change from cloning and is something to do while I wait for new ligase to arrive.

Ligase is the enzyme that is supposed to stick my new favourite piece of DNA into the green-fluorescent protein vector so that it forms a circle again. This circle is then what I introduce to the bacterial cells in the hope that they'll take it up and make more of it (like little microbiological factories). Then, when they've made more of it, I extract the circular DNA (but not the bacteria's chromosomal DNA) back from the bugs and transfect it into the liver cells I have going in culture. Then I get to treat those cells with compounds of interest to see whether my favourite protein will bind to it and take it into the nucleus of the cell (the GFP tag means you can see where the proteins are (mostly) in the cell.

None of this has a chance of working if the ligase won't stick the DNA back together. I'm hoping (really, really hoping) new ligase will solve all my problems.

In the meantime, I have a ton of modelling to catch up on.
24th Nov 2009, 01:32   | tags:comments (4)

Cloning fail. Again.

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:(
22nd Nov 2009, 20:10   | tags:comments (1)

Transformations

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Really hope this works. Took nearly a full week to re-do all the cloning. I've done every step as best I can, checking along the way. Tomorrow I'll know whether it was worth it or whether I'll be needing more drawing boards.

Really, really hope this works.
21st Nov 2009, 19:16   | tags:comments (2)

These are not the clones you are looking for

(viewed 356 times)
After 6 days a very few tiny, tiny colonies have appeared on my plates. I don't trust them. Cells transformed with control plasmids produced really good colonies overnight so however these ones are resisting the antibiotic, I suspect, is not down to me. I will test them next week, but for now I'll carry on with the re-do I'm nearly at the end of.

A new protein modelling program for me to learn how to use, and hopefully use to sort out all the 'improper' bond angles, side-chain clashes etc of my other favourite protein, to get it into a fit state for docking ligands into. That's the hope at least. How far can I get without having to really, really get into the nuts and bolts of molecular dynamics? I'm hoping quite far. I have to hope. Then I have to read. A lot.

Another day, another gel, ligations set up over night again. Tomorrow will be the day of transformations and Sunday I will find out if any of this week has been worth the effort.
21st Nov 2009, 01:56   comments (0)