But anyway, here's my week.
On Monday, I've been continuing to test this one primer on a whole bunch of DNA plates. The primer is called up1 which is upstream from this certain shattering gene that should be present in O. barthii and not in O. glaberrima. If you remember, O. barthii is a wild type rice from Western Africa and O. glaberrima is a domesticated type that seems to have descended from O. barthii. (I think I talked about it in Week 2.) Also remember that the domesticated rice have evolved to reduce shattering (some papers called this trait among others the domestication syndrome) so they will most likely not have the shattering gene.
But anyway, I've been assigned to test this primer by making PCRs and gels with this primer. So, I made a gel of a PCR that I did last week which was labeled "plate 2" and did a PCR of another plate called "plate 4." So, each plate has 96 wells (12 x 8) and is filled with DNA. I'm not sure about my mentor and another member of the AGI team who has been helping me tons (his name is Dario), but I don't know which species are in which well or even which plate. All I know is that there are three options: barthii, glaberrima, or it's heterozygous. That's what the gels from gel electrophoresis tells us. (I'll talk about it a little more as you scroll down).
Continuing on, I also got to design some new primers with Dario. He showed me 4 websites that are all related to creating primers. I only remember two of them which are called Primer3Plus and the NCBI BLAST. I'm sure you have ran into PubMed once or twice while researching (especially bio people). Well, PubMed is a resource from the National Center for Biotechnology Information (NCBI), and NCBI BLAST is another. The NCBI BLAST is pretty fun to play around. It provides gene maps of certain species and chromosomes and when used with another website that has a similar function to this websites, they can provide sequences. Then you plug in this sequence into the Primer3Plus and they find a forward and reverse primer for you with the certain conditions that you want them in. They give you multiple choices so you can choose from them. An important thing you want to look for in primers are if the forward and reverse primers have near the same temperature, whether they repeat with themselves (match with themselves), whether they match with each other. Another good thing is if the primer ends have a cytosine (C) or guanine (G) base because these bases make three hydrogen bonds with each other while adenine (A) and thymine (T) make two hydrogen bonds making them less secure. With a C or G at the ends, you feel more reassured that the primers have been bound to the DNA so that the Taq polymerase can do the rest. You also want to make sure that the primers don't repeat with themselves or each other because that could create a primer cloud in the gel which is when the primers do bind with themselves and also create a small faint fragment of DNA that travels down in the gel. Once you find the primers, you then insert the primers into the NCBI BLAST to make sure that the primers are unique to the sequence that you are looking at and won't bind to other parts of the species such as at a different chromosome or near a different gene. Once you verify that, you can order them. We're still waiting on those orders.
On Wednesday, I made another gel of another plate that I did the previous week. Then I went into a lab meeting. One member from the AGI team did a 15 minute talk on some of the data that he ran with Dario. He was showing also how he used R (also in Week 2) and he used this program called "vioplot" or "violin plot." This plot creates a box-and-whisker plot and with another graph that seems like a line graph, but the line graph is creates on both sides of the box-and-whisker plot so that it is symmetrical and creates a violin shape. Also, my advisor Dave led the rest of the lab meeting to talk about the new sequencing project. They're are planning to sequence two types of rice that are both under the O. sativa ssp. indica group called Minghua36 and Zhenshan97. For now, they're going to make lots of BACs and then use this new machine from PacBio and use this method called SMRT (single molecule real time sequencing). This method was kind of confusing for me and it seems like there's going to be some training on how to use the machine and do all the sequencing, but it looked pretty cool.
While waiting for my gel to finish, I ran more PCRs on plate 7 and another plate called the reextraction plate where Dario and my mentor Naomi re-extracted DNA from certain species that seemed to be missing information from the other plates.
I also did some scoring of the gels with Dario. Scoring is where I'll tell you more about the gels and which is what species. Right now, I am basically trying to determine where and why O. glaberrima does not have the shattering gene and where and why O. barthii does have the shattering gene. In order to do that, I have to genotype the DNA plates that I have been working on and I do that by running gels and scoring them. We have tested the up1 primer with a known O. glaberrima, O. barthii, and a known heterozygous, so we know how to determine which bands belongs to which species. (Coincidentally, William George actually did this...he was doing a research internship thing over the summer).
On Friday, I did some DNA extraction with my mentor Naomi. We extracted DNA from rice leaves and awns (which are little brittle hair-like things that stick out near the grains). I realized that DNA extraction was a really long process and has to do with A LOT of waiting. After extracting the DNA, we Nanodrop-ed each sample. The Nanodrop is a machine that measures the concentration and absorption (of light) of DNA. Most people don't like using the Nanodrop because it seems like it just spits out random numbers so we also run a gel of the samples.
Also during one of the waiting times, I ran a gel of the plate 7 and the reextraction plate where I did the PCRs Wednesday.
Well that was my week. Research-wise, I've been able to find some sources on shattering genes mostly on sh1 and sh4. A lot of research has been done on O. sativa and its subspecies but not much on African rice. I'm still trying to find some more sources. I did find an article with the title, "Oryza glaberrima: A source for the improvement of Oryza sativa" which seems promising. I'll finish this in a couple days while looking for more sources.
I also forgot to mention that I got a T-shirt last week. (I mean, who doesn't love a free T-shirt?) Every member of the AGI team gets one, and it's related to the project and the 9 billion people question (refer to Week 1 if you haven't heard about it.)
Feel free to leave any comments or questions! Have a great spring break!!
But anyway, I've been assigned to test this primer by making PCRs and gels with this primer. So, I made a gel of a PCR that I did last week which was labeled "plate 2" and did a PCR of another plate called "plate 4." So, each plate has 96 wells (12 x 8) and is filled with DNA. I'm not sure about my mentor and another member of the AGI team who has been helping me tons (his name is Dario), but I don't know which species are in which well or even which plate. All I know is that there are three options: barthii, glaberrima, or it's heterozygous. That's what the gels from gel electrophoresis tells us. (I'll talk about it a little more as you scroll down).
Continuing on, I also got to design some new primers with Dario. He showed me 4 websites that are all related to creating primers. I only remember two of them which are called Primer3Plus and the NCBI BLAST. I'm sure you have ran into PubMed once or twice while researching (especially bio people). Well, PubMed is a resource from the National Center for Biotechnology Information (NCBI), and NCBI BLAST is another. The NCBI BLAST is pretty fun to play around. It provides gene maps of certain species and chromosomes and when used with another website that has a similar function to this websites, they can provide sequences. Then you plug in this sequence into the Primer3Plus and they find a forward and reverse primer for you with the certain conditions that you want them in. They give you multiple choices so you can choose from them. An important thing you want to look for in primers are if the forward and reverse primers have near the same temperature, whether they repeat with themselves (match with themselves), whether they match with each other. Another good thing is if the primer ends have a cytosine (C) or guanine (G) base because these bases make three hydrogen bonds with each other while adenine (A) and thymine (T) make two hydrogen bonds making them less secure. With a C or G at the ends, you feel more reassured that the primers have been bound to the DNA so that the Taq polymerase can do the rest. You also want to make sure that the primers don't repeat with themselves or each other because that could create a primer cloud in the gel which is when the primers do bind with themselves and also create a small faint fragment of DNA that travels down in the gel. Once you find the primers, you then insert the primers into the NCBI BLAST to make sure that the primers are unique to the sequence that you are looking at and won't bind to other parts of the species such as at a different chromosome or near a different gene. Once you verify that, you can order them. We're still waiting on those orders.
On Wednesday, I made another gel of another plate that I did the previous week. Then I went into a lab meeting. One member from the AGI team did a 15 minute talk on some of the data that he ran with Dario. He was showing also how he used R (also in Week 2) and he used this program called "vioplot" or "violin plot." This plot creates a box-and-whisker plot and with another graph that seems like a line graph, but the line graph is creates on both sides of the box-and-whisker plot so that it is symmetrical and creates a violin shape. Also, my advisor Dave led the rest of the lab meeting to talk about the new sequencing project. They're are planning to sequence two types of rice that are both under the O. sativa ssp. indica group called Minghua36 and Zhenshan97. For now, they're going to make lots of BACs and then use this new machine from PacBio and use this method called SMRT (single molecule real time sequencing). This method was kind of confusing for me and it seems like there's going to be some training on how to use the machine and do all the sequencing, but it looked pretty cool.
While waiting for my gel to finish, I ran more PCRs on plate 7 and another plate called the reextraction plate where Dario and my mentor Naomi re-extracted DNA from certain species that seemed to be missing information from the other plates.
I also did some scoring of the gels with Dario. Scoring is where I'll tell you more about the gels and which is what species. Right now, I am basically trying to determine where and why O. glaberrima does not have the shattering gene and where and why O. barthii does have the shattering gene. In order to do that, I have to genotype the DNA plates that I have been working on and I do that by running gels and scoring them. We have tested the up1 primer with a known O. glaberrima, O. barthii, and a known heterozygous, so we know how to determine which bands belongs to which species. (Coincidentally, William George actually did this...he was doing a research internship thing over the summer).
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| The picture of the gel that we refer to when we're scoring the other gels. |
Also during one of the waiting times, I ran a gel of the plate 7 and the reextraction plate where I did the PCRs Wednesday.
Well that was my week. Research-wise, I've been able to find some sources on shattering genes mostly on sh1 and sh4. A lot of research has been done on O. sativa and its subspecies but not much on African rice. I'm still trying to find some more sources. I did find an article with the title, "Oryza glaberrima: A source for the improvement of Oryza sativa" which seems promising. I'll finish this in a couple days while looking for more sources.
I also forgot to mention that I got a T-shirt last week. (I mean, who doesn't love a free T-shirt?) Every member of the AGI team gets one, and it's related to the project and the 9 billion people question (refer to Week 1 if you haven't heard about it.)
 |
| Sorry, I couldn't figure out why it rotated. But it says, "rice is life." |
Brittnay, is it by design that you don't know which species' DNA is in each well? A blind test?
ReplyDeleteI think it might be partially. But someone has to have made the DNA plates and all of the DNA has been extracted from rice plants growing at a greenhouse at the U of A. In our excel where we do our scoring, I saw that my mentor and Dario recorded the plants that correlate with each well, so my guess is that they might know what species the DNA belongs to and we're just making sure by testing the DNA with multiple primers to make sure of the genotypes. We test primers that are upstream, downstream, and somewhere in the middle of the gene to make sure which species' DNA it is. Normally, they should all line up and have the same genotype, but if they don't, we try to test them again and sometimes, if the downstream primers show a different genotype, it is possible that there may have been some recombination.
DeleteBut in all, I don't think it is completely a blind test. I think we're really just testing out hypotheses.