laurha
bands
These bands tell me what’s inside the genes of the plants I’m growing, isn’t it awesome?
I’m going to try to explain it (just to practice my written english):
I cut one leaf of each one of “my plants”, and put them inside different tubes. Then I grind and “cook” the leaf with some liquids, following a simple recipe, to extract the DNA. At the end (less than 1 hour with 40 samples and I’m still quite slow), I obtain a white gelatinous stuff in tubes, the DNA of each leaf.
Then I place a drop of this DNA (0,2 μL, nothing!!) that belongs to each leaf in small PCR-tubes with a “reaction mix” that contains an enzyme able to make DNA copies (borrowed from a bacteria that lives in hot spots), and something called “primers”, let's explain what they are:
As everybody knows, DNA is a double helix. In order to make a copy, both strands have to be separated (e.g. by heating), and then the enzyme copy every single strand in a way that it results in a new double helix, with the old strand used as a mold and a 'brand new strand'.
But the enzyme can’t just start copying a strand without a small amount of 'double helix sequence' already conformed, that’s why we need the primers. They are short sequences of “single strand” that complements (and therefore binds to) a specific region in the genome (the one you want to gossip). As they bind, they conform a short double strand at this point, and once the enzyme has this “starting point” is able to makes copies and copies and copies of that precise DNA region.
It’s necessary to heat and cool the reaction mix for every copy-cycle, so I place the tubes (with the DNA, the enzyme, the primers and the molecules the enzyme needs to build the DNA sequence) in a machine that heats&cools the tubes in cycles. In 2,5 hours (40 cycles) I get a few drops (20 µl) with thousands of copies of the sequence chosen by the primers and amplified by the enzyme.
The next step is to find out what’s inside the gene I’m interested in, and the easiest way to do it is: look at the size!! If the amplified sequence has the right size, there’s nothing weird in that plant, it’s “wildtype”. But we work with mutant plants bearing insertions on the genes that makes them useless to do their job (this is, to build proteins). So, if the sequence is too long (or just if there’s just not amplified sequence because the enzyme can’t copy a very long sequence) that means an insertion inside, hence this plant is a “knockout” for this gene. In other words, this gene doesn’t work in this plant.
How do I find out the length? easy: I pour the liquid with the amplified sequence inside a gel (each sample inside a different well) and apply an electric field. The DNA starts literally running towards the + electrode (because the DNA molecule has negative charge), but the bigger the fragment the slower it will move, due to the density of the gel. After less than half an hour, fragments with different lengths form bands at different levels. This is the moment to take a beautiful picture.
My goal now is to get a single plant with 2 knockout genes. In order to obtain the "double mutant", we cross 2 plants, each one with a single mutation, and wait for the seeds to grow… but, who knows which one of the children inherited both mutations? just cutting a single leaf and looking at the length of these amplified sequences.
You can see the result in the b/w picture with rows of bands over my lab-book. Each row belongs to a leaf (of a single plant), and each band to an amplified sequence. Depending of the position of the bands, I know the length of the sequence and therefore what’s happening inside of "her" DNA.
And everything in one single morning, definitely awesome!
PS. Thanks to my reviewers for helping me so much!!
bands
These bands tell me what’s inside the genes of the plants I’m growing, isn’t it awesome?
I’m going to try to explain it (just to practice my written english):
I cut one leaf of each one of “my plants”, and put them inside different tubes. Then I grind and “cook” the leaf with some liquids, following a simple recipe, to extract the DNA. At the end (less than 1 hour with 40 samples and I’m still quite slow), I obtain a white gelatinous stuff in tubes, the DNA of each leaf.
Then I place a drop of this DNA (0,2 μL, nothing!!) that belongs to each leaf in small PCR-tubes with a “reaction mix” that contains an enzyme able to make DNA copies (borrowed from a bacteria that lives in hot spots), and something called “primers”, let's explain what they are:
As everybody knows, DNA is a double helix. In order to make a copy, both strands have to be separated (e.g. by heating), and then the enzyme copy every single strand in a way that it results in a new double helix, with the old strand used as a mold and a 'brand new strand'.
But the enzyme can’t just start copying a strand without a small amount of 'double helix sequence' already conformed, that’s why we need the primers. They are short sequences of “single strand” that complements (and therefore binds to) a specific region in the genome (the one you want to gossip). As they bind, they conform a short double strand at this point, and once the enzyme has this “starting point” is able to makes copies and copies and copies of that precise DNA region.
It’s necessary to heat and cool the reaction mix for every copy-cycle, so I place the tubes (with the DNA, the enzyme, the primers and the molecules the enzyme needs to build the DNA sequence) in a machine that heats&cools the tubes in cycles. In 2,5 hours (40 cycles) I get a few drops (20 µl) with thousands of copies of the sequence chosen by the primers and amplified by the enzyme.
The next step is to find out what’s inside the gene I’m interested in, and the easiest way to do it is: look at the size!! If the amplified sequence has the right size, there’s nothing weird in that plant, it’s “wildtype”. But we work with mutant plants bearing insertions on the genes that makes them useless to do their job (this is, to build proteins). So, if the sequence is too long (or just if there’s just not amplified sequence because the enzyme can’t copy a very long sequence) that means an insertion inside, hence this plant is a “knockout” for this gene. In other words, this gene doesn’t work in this plant.
How do I find out the length? easy: I pour the liquid with the amplified sequence inside a gel (each sample inside a different well) and apply an electric field. The DNA starts literally running towards the + electrode (because the DNA molecule has negative charge), but the bigger the fragment the slower it will move, due to the density of the gel. After less than half an hour, fragments with different lengths form bands at different levels. This is the moment to take a beautiful picture.
My goal now is to get a single plant with 2 knockout genes. In order to obtain the "double mutant", we cross 2 plants, each one with a single mutation, and wait for the seeds to grow… but, who knows which one of the children inherited both mutations? just cutting a single leaf and looking at the length of these amplified sequences.
You can see the result in the b/w picture with rows of bands over my lab-book. Each row belongs to a leaf (of a single plant), and each band to an amplified sequence. Depending of the position of the bands, I know the length of the sequence and therefore what’s happening inside of "her" DNA.
And everything in one single morning, definitely awesome!
PS. Thanks to my reviewers for helping me so much!!