Hey guys. So everything has been winding down with our SRPs. I've been working on my presentation and still trying to wrap my head around my paper. While everyone seems to be done with their internships, I have still been going to mine. I was given the opportunity to continue working with the AGI team until the end of May (after all the SRP stuff) which is really great since I get more experience in the lab. I may even begin helping with their new PacBio project on sequencing two different rice varieties before I get into my summer vacation.
Lately, I've been catching up on some reading to see if there's anything more relevant I can add into my presentation background-wise. I also have been questioning about my researched lab report as my final product. Although I have been testing out primers on certain shattering genes, we haven't been able to make conclusive results, so I don't think I'll be able to add in a "results" section at all. Also, we have been working on primers on shattering genes that greatly affected the domestication of Oryza sativa called sh4 and qsh1 but they haven't been working. Sh4 is a quantitative trait locus (QTL) on chromosome 4 in rice and qsh1 is a QTL on chromosome 1 in rice. From different experiments, both of these primers explained 69% of the phenotypic variance between shattering and non-shattering. Currently, I am designing new primers on these genes but won't be able to test them until after my SRP presentation, so I won't get any results until then.
I am possibly thinking of continuing my paper as just a research paper and maybe including some of my experiences in it. But, I will be talking to my advisors about it (hopefully today).
Monday, April 21, 2014
Monday, April 7, 2014
Week 9
Week 9! I can't believe it's almost over!
This week was pretty quiet. I haven't been going to the lab as much. I've been mainly doing to EFE repeat characterization with Dario that I mentioned last week. We should be finishing up soon. I also did a PCR with some primer combinations on a qSh1 gene, which is related to shattering. However, when I tested this last time, the bands in the gels were the same length which shouldn't happen since it seems that O. glaberrima has an insertion in this qSh1 gene of about 100 base pairs, so there should be two bands of different lengths, about 100 bp apart. Sadly, we don't see that, so we may have the rest of the PCR products and sequence them.
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| gel of the qSh1 combos |
I also did a PCR and ran a gel on Friday (mostly because I was bored from staring at my computer screen doing EFE characterizations). I tested a primer that was not linked to a shattering gene and was successful in showing two different bands in the parents (barthii and glaberrima). This primer was on chromosome 8 in O. barthii but since the two parents are related, the primers should also attach to the same region in O. glaberrima. I tested this primer on some DNA plates like I did with the up1 primer few weeks back.
I also started my outline for my final project. It's going to be more of a lab report even though I don't have a lot of conclusive results since this shattering project isn't completely over. I hope to finish up my outline, look for some more sources, and at least start on my introduction by end of next week.
Monday, March 31, 2014
Week 8:
Hey guys!
This week was basically the same as all my other weeks. Tons of PCRs and gels. Lately, I've been re-doing PCRs and gels of primers that we have done in the past but haven't gotten nice results. I also re-tested the awns where we extracted DNA from a few weeks ago. Unfortunately, we haven't gotten good results because the awns aren't concentrated enough. One solution we have in mind for now is to re-suspend the DNA pellet, centrifuge, and test their concentrations again.
On Wednesday, I went into a lab meeting. This meeting was basically an introduction to the new PacBio machine that came in couple weeks ago. An employee from the PacBio company came in and talked about how to use the machine, the prep, what to do if anything went wrong (mostly it's to contact the PacBio tech support or the guy that came in), and how to analyze the results. After the meeting, I began scoring and starting to organize the genotypes of the species.
On Friday, it was something different. I didn't go into the lab at all which was kind of strange for me since I've been in the lab basically since day 1 of my internship. I actually got to do some bioinformatics stuff. It's related to rice but I don't think it's quite related to what I'm doing. I've been looking at medium-sized chunks of repeats gathered and organized by Dario and his ____ in Australia. These chunks contain one or both of two different type of viruses that have been present in other plants close to rice such as wheat. So for now, I have been looking at the chunks that are recorded to be present in one rice genome and comparing them to other rice genomes to see if they are present. Basically, I'm looking for polymorphisms even though I haven't seen the different phenotypes. The tools I have been using are these sites called Genome Browser. There are two sites where one helps to track the location of the repeat chunk and nearby genes while the other site helps to compare a certain site (normally the repeat along with at least 2 nearby genes) and compare it to other rice genomes. I found out that it was important to have at least one nearby gene in the window of the chromosome that I was looking at when I was comparing the region with other genomes because if you just have the repeat, it could be present all over another rice genome but in different chromosomes (since it's a repeated sequence). With a gene in the region, it helps to anchor the repeat in the other genomes, if present. Also, most likely when they are present, they should be present in an area on the same chromosome and same area as the first, or what the site calls the reference, genome.
And that was my week. It's getting so close to the end. So this next week, I'll be continuing my lab work and start organizing the information I've gathered and the data I've collected to start working on my final product which is going to be a research paper. Sadly, I haven't done much bioinformatics stuff or analyzed the data so it won't have as many numbers as I had hoped, so no p-value or chi-squared or anything like that sadly. I'll also be looking for more sources.
So that's it for now. Bye!
This week was basically the same as all my other weeks. Tons of PCRs and gels. Lately, I've been re-doing PCRs and gels of primers that we have done in the past but haven't gotten nice results. I also re-tested the awns where we extracted DNA from a few weeks ago. Unfortunately, we haven't gotten good results because the awns aren't concentrated enough. One solution we have in mind for now is to re-suspend the DNA pellet, centrifuge, and test their concentrations again.
On Wednesday, I went into a lab meeting. This meeting was basically an introduction to the new PacBio machine that came in couple weeks ago. An employee from the PacBio company came in and talked about how to use the machine, the prep, what to do if anything went wrong (mostly it's to contact the PacBio tech support or the guy that came in), and how to analyze the results. After the meeting, I began scoring and starting to organize the genotypes of the species.
On Friday, it was something different. I didn't go into the lab at all which was kind of strange for me since I've been in the lab basically since day 1 of my internship. I actually got to do some bioinformatics stuff. It's related to rice but I don't think it's quite related to what I'm doing. I've been looking at medium-sized chunks of repeats gathered and organized by Dario and his ____ in Australia. These chunks contain one or both of two different type of viruses that have been present in other plants close to rice such as wheat. So for now, I have been looking at the chunks that are recorded to be present in one rice genome and comparing them to other rice genomes to see if they are present. Basically, I'm looking for polymorphisms even though I haven't seen the different phenotypes. The tools I have been using are these sites called Genome Browser. There are two sites where one helps to track the location of the repeat chunk and nearby genes while the other site helps to compare a certain site (normally the repeat along with at least 2 nearby genes) and compare it to other rice genomes. I found out that it was important to have at least one nearby gene in the window of the chromosome that I was looking at when I was comparing the region with other genomes because if you just have the repeat, it could be present all over another rice genome but in different chromosomes (since it's a repeated sequence). With a gene in the region, it helps to anchor the repeat in the other genomes, if present. Also, most likely when they are present, they should be present in an area on the same chromosome and same area as the first, or what the site calls the reference, genome.
And that was my week. It's getting so close to the end. So this next week, I'll be continuing my lab work and start organizing the information I've gathered and the data I've collected to start working on my final product which is going to be a research paper. Sadly, I haven't done much bioinformatics stuff or analyzed the data so it won't have as many numbers as I had hoped, so no p-value or chi-squared or anything like that sadly. I'll also be looking for more sources.
So that's it for now. Bye!
Wednesday, March 26, 2014
Week 7:
Hi guys! I'm so sorry for the late post! Hope you guys have been enjoying your internships and has a great spring break. It's already past week 7!
Well anyways, here's what I've been up to. Even though it was Spring Break last week, I still had to go to my internship. On Monday, we finally received the new primers that Dario and I designed the week before. I diluted the new primers and prepared the forward and reverse primer mixes. Then, I prepared for a PCR on the new primers with just the O. glaberrima parent (we ran out of O.barthii and the heterozygous parents).
On Wednesday, I ran a gel of the PCR reaction from Monday. Unfortunately, most of the primer combinations did not work, and most of them were the sh4 primers which are linked to the sh4 gene related to shattering. This gene is said to be involved in the development of the abscission layer which is a layer of cells that disintegrates leading the grains to fall off, or shatter. Of the 5 out of 15 primer combinations that did work, we prepared a new PCR with a new condition where we did a 40 second extension step instead of a minute and 20 seconds at 72 degrees Celsius.
I also finished reading the article from last week. Basically, the article talks about the good traits of O. glaberrima such as weed competitiveness, drought resistance, and tolerance for abiotic stresses such as acidic conditions and iron and aluminum toxicity. If combined with O.sativa and its high-yielding traits then we can produce a new kind of rice called NERICA (NEw RIce for AfriCA). However, there some interspecific incompatibility such as sterilization. There are still many questions on this that are unanswered.
Finally, on Friday, I did a PCR and a gel on a DNA plate with the down1 primer which is downstream from the focus target of the gene. Also, I had to make a nice (meaning really pretty and clear) gel of the 5 primer combinations that amplified earlier that week. Sadly, most of the bands were hard to distinguish since the O. glaberrima and the O. barthii parents were so close together. Even the known-heterozygous parent (which is just a mixture of glaberrima and barthii) had only one band when there should be two. After looking at the results, Dario and I concluded that we'll just have to design more new primers later.
Also, the PacBio (Pacific Biosciences) came in last week. They set up and performed some test runs. It seems like they're going to start using it this week.
Well, here's my Week 7! Sorry for the late post. I'll post up my Week 8 at the end of this week. Until then...See ya! Hope you guys have another great week!
Well anyways, here's what I've been up to. Even though it was Spring Break last week, I still had to go to my internship. On Monday, we finally received the new primers that Dario and I designed the week before. I diluted the new primers and prepared the forward and reverse primer mixes. Then, I prepared for a PCR on the new primers with just the O. glaberrima parent (we ran out of O.barthii and the heterozygous parents).
On Wednesday, I ran a gel of the PCR reaction from Monday. Unfortunately, most of the primer combinations did not work, and most of them were the sh4 primers which are linked to the sh4 gene related to shattering. This gene is said to be involved in the development of the abscission layer which is a layer of cells that disintegrates leading the grains to fall off, or shatter. Of the 5 out of 15 primer combinations that did work, we prepared a new PCR with a new condition where we did a 40 second extension step instead of a minute and 20 seconds at 72 degrees Celsius.
I also finished reading the article from last week. Basically, the article talks about the good traits of O. glaberrima such as weed competitiveness, drought resistance, and tolerance for abiotic stresses such as acidic conditions and iron and aluminum toxicity. If combined with O.sativa and its high-yielding traits then we can produce a new kind of rice called NERICA (NEw RIce for AfriCA). However, there some interspecific incompatibility such as sterilization. There are still many questions on this that are unanswered.
Finally, on Friday, I did a PCR and a gel on a DNA plate with the down1 primer which is downstream from the focus target of the gene. Also, I had to make a nice (meaning really pretty and clear) gel of the 5 primer combinations that amplified earlier that week. Sadly, most of the bands were hard to distinguish since the O. glaberrima and the O. barthii parents were so close together. Even the known-heterozygous parent (which is just a mixture of glaberrima and barthii) had only one band when there should be two. After looking at the results, Dario and I concluded that we'll just have to design more new primers later.
Also, the PacBio (Pacific Biosciences) came in last week. They set up and performed some test runs. It seems like they're going to start using it this week.
Well, here's my Week 7! Sorry for the late post. I'll post up my Week 8 at the end of this week. Until then...See ya! Hope you guys have another great week!
Monday, March 17, 2014
Week 6:
I think we're already at the halfway point in our SRPs. It's so crazy how much time has already passed by.
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.)
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| Sorry, I couldn't figure out why it rotated. But it says, "rice is life." |
Monday, March 10, 2014
Week 5
Hey guys! Hope you're internships are going well. Here's my week!
My internship has been pretty routine. I come and make PCRs and run gels, and that's basically what I did this week also.
On Monday, I ran a gel testing a primer called upstream 1 (up1 for short) with a row of DNA from a DNA plate. The results were much better than what we had tested before, and the only difference was that, with trial and error, I changed the annealing temperature in the PCR machine from 58 degrees Celsius to 55 degrees. It makes a world of difference! Look for yourself.
I used exactly the same reagants which were the DNA, up1 primer, GoTaq (which is the Taq polymerase that makes the other strand of DNA by binding nitrogenous bases using the base pair rule after denaturing at 94 degrees Celsius). Seeing how this experiment worked and seeing that the DNA sufficiently amplified in the PCR, I started to do this with other plates of DNA using the same conditions.
For this gel, the PCR used the same up1 primer and same conditions in the PCR machine. The only thing different was that I used a different DNA plate. I was so happy to get such great results!
My internship has been pretty routine. I come and make PCRs and run gels, and that's basically what I did this week also.
On Monday, I ran a gel testing a primer called upstream 1 (up1 for short) with a row of DNA from a DNA plate. The results were much better than what we had tested before, and the only difference was that, with trial and error, I changed the annealing temperature in the PCR machine from 58 degrees Celsius to 55 degrees. It makes a world of difference! Look for yourself.
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| Gel with PCR at annealing temperature of 58 degrees Celsius |
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| Gel with PCR at annealing temperature of 55 degrees Celsius (same reagants) |
On Wednesday, I went into a lab meeting. This week, a guy from the AGI team just talked about what he has been doing lately and some of his results. He mainly dealt with the construction of the BAC library (bacterial artificial chromosome - which is a DNA construct used for transforming and cloning in bacteria, usually E.coli and is often used the sequence genomes) for the Oryza (rice) project. He also mentioned a CHEF gel which I have never heard of and shears (I think that's what they're called), but when I saw pictures of his gels, they looked really cool. Instead of looking like the thin bands above, they was a large chunk in each column. Instead of giving a specific size such as the thin bands above, the results of these shears provide more of a range. But I liked it when he was talking about his methods, and the team was discussing whether these methods were sufficient enough in purifying the DNA when making the BAC library and also what to do next and compare the results.
After the lab meeting, I made a large gel of the PCR that I made on Friday. Here's a picture!
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| Large gel (GREAT RESULTS!) |
On Friday, I did another PCR with more DNA plates using the up1 primer and ran some gels. However, I also got to grind some leaves from rice plants, so that we could do DNA extractions the next week. We froze the leaves in liquid nitrogen which was fun. Afterwards, I ground some awns that my mentor collected a while back.
Well that was my week. Hope you guys are doing well on your internships! See you next time :)
Monday, March 3, 2014
Week 4
Hello. I can't believe it's already the beginning of March!
I've enjoyed reading all the posts that you guys have put up. All of these internships sound so cool and so interesting. Now, here's my week. This week was actually pretty busy. I feel that my internship has been preparing me for the real world; I even work practically full hours (8AM-5PM) 3 days a week...and soon maybe even everyday. I am going to take part in another project, not related to my SRP, that has to do with sequencing some genomes with a new method. Although it isn't a part of my SRP, I think it will be a good experience to have.
Monday: I ran some gels with my mentor Naomi. Lately we have been re-testing old data and also some new primers to see if they are amplifying correctly. I also ran a PCR to test some of the primers again.
Tuesday: I finally took my biosafety protection course and I passed. Now, I can officially work in the lab (even though I already have been under supervision) still with supervision but a little bit more independently.
Wednesday: This day went by really fast and was packed with a whole bunch of stuff although it may not really sound like it. My mentor and I both came in a little late so we only had time to make a gel before going into the weekly lab meetings. For this lab meeting, Dr. Yu (who is also a high school friend of my dad) gave the talk on his data with Tandem Array Genes (TAGs). Although he explained exactly what they are, I still have trouble understanding. What I understood is that these TAGs are take up a large proportion of genomes and they are a reservoir of genetic redundancy. I will look into the purposes of these TAGs and how they are useful so tune in next week. I also made some 100 base-pair (bp) ladders that are used in the gels but no one seemed to know exactly how to make it so we experimented a mixture and tested it in a gel along with some samples. There was also a power outage in the middle of the day (apparently it happens more often than you would think) but good thing there's a back-up generator since not everything turned off. I also learned to disassemble and clean pipettes and reassemble them. A dirty pipette may be the reason why it cannot pipette the exact amount correctly which could lead to inaccuracy in an experiment (and we wouldn't want that, right?)
Friday: I ran another PCR testing one of the samples that I ran on Wednesday. Then, I made a gel and ran the PCR samples that my mentor Naomi made on Thursday. I also cleaned some more pipettes and made some more 100 bp ladders.
Well, that was my week. Hope you guys have another great week! Bye~~
I've enjoyed reading all the posts that you guys have put up. All of these internships sound so cool and so interesting. Now, here's my week. This week was actually pretty busy. I feel that my internship has been preparing me for the real world; I even work practically full hours (8AM-5PM) 3 days a week...and soon maybe even everyday. I am going to take part in another project, not related to my SRP, that has to do with sequencing some genomes with a new method. Although it isn't a part of my SRP, I think it will be a good experience to have.
Monday: I ran some gels with my mentor Naomi. Lately we have been re-testing old data and also some new primers to see if they are amplifying correctly. I also ran a PCR to test some of the primers again.
Tuesday: I finally took my biosafety protection course and I passed. Now, I can officially work in the lab (even though I already have been under supervision) still with supervision but a little bit more independently.
Wednesday: This day went by really fast and was packed with a whole bunch of stuff although it may not really sound like it. My mentor and I both came in a little late so we only had time to make a gel before going into the weekly lab meetings. For this lab meeting, Dr. Yu (who is also a high school friend of my dad) gave the talk on his data with Tandem Array Genes (TAGs). Although he explained exactly what they are, I still have trouble understanding. What I understood is that these TAGs are take up a large proportion of genomes and they are a reservoir of genetic redundancy. I will look into the purposes of these TAGs and how they are useful so tune in next week. I also made some 100 base-pair (bp) ladders that are used in the gels but no one seemed to know exactly how to make it so we experimented a mixture and tested it in a gel along with some samples. There was also a power outage in the middle of the day (apparently it happens more often than you would think) but good thing there's a back-up generator since not everything turned off. I also learned to disassemble and clean pipettes and reassemble them. A dirty pipette may be the reason why it cannot pipette the exact amount correctly which could lead to inaccuracy in an experiment (and we wouldn't want that, right?)
Friday: I ran another PCR testing one of the samples that I ran on Wednesday. Then, I made a gel and ran the PCR samples that my mentor Naomi made on Thursday. I also cleaned some more pipettes and made some more 100 bp ladders.
Well, that was my week. Hope you guys have another great week! Bye~~
Monday, February 24, 2014
Week 3
Hello again! I hope you guys have been enjoying your internships and your Rodeo Break.
On Monday, Feb. 17th, I started to run gels with my mentor Naomi. We ran a small gel with a primer called up2 which was a primer that is a little before the target segment of DNA that we're looking at. Later, we scored some older gels that the AGI team made and took pictures of and decided to re-run some of them. So we ran large gels with PCR plates that were made before. I got to insert the DNA using a 12 channel multi-channel pipette. At first, it got a little tricky trying to make sure I got all the PCR products inside each pipette and that I put all of the product inside each well and the pipette kind of gets unbalanced as you pipette out the PCR products. So I also did some practice pipetting with water and a plate.
After seeing the results of the gels we ran, we decided that the primers that were used before may not be working such as the down1 primer (which is further down the DNA from the target segment). The bands seen in the gel represent whether the DNA have the O. glaberrima or O. barthii allele or both. Also, the ends of each gel are the base pair ladders (I believe for this gel we used the 100 bp ladder). You could also see how there were some gaps in the gel...there might not have been DNA in there.
Therefore, I got to prepare new primer solutions. Mostly, it consisted of making tons of dilutions and dealing not only with molarity but also nano-moles and micro-molar solutions. I felt like it was chemistry math (mainly the dilution equation: M1V1 = M2V2) on a whole different (smaller) level. These new primers were called qSh1-1, qSh1-2, sh4-b1, sh4-b2, sh4-b3-R. So instead of just handing out the primers already made for us like in our Capstone Bio class with the mitochondrial DNA primers, I got to actually prepare the forward and reverse primer solutions. Although it was just simply diluting and pipetting, I'm glad I got to know how things were made from the beginning.
On Wednesday, I observed my mentor preparing the PCR with the new primer solutions I made. After loading the PCR mixes into the PCR machine, I went into my second lab meeting. The more I see these lab meetings, the more I think, "Good thing we got lots of practice presenting during our capstone classes." Although these lab meetings are more of discussions and informal presentations, I could notice things that went well and things that didn't. But anyway, the main topic for this lab meeting was on new data relating to SNPs and imputations. I'm not sure what those terms mean and I think the other members of the AGI team didn't quite understand either, so a lot of time was spent trying to understand how the data was computed. At the end of the meeting, there was a discussion about rice and arsenic (which thankfully I had some background info in). It seemed as though there was more arsenic in the food supply in general than what everybody thought. Although there were jokes about how this fact might negatively affect the rice project the AGI team was doing (since they said rice was the answer to the 9-billion people question and could feed the world). However, it also seemed like the arsenic was coming into the food supply not only from natural causes such as volcano eruptions but also from human activities such as using pesticides/herbicides, mining, and burning coal and oil. So there were also discussions of how this issue of rice and arsenic can also provide a new path for research since the team was already interested in starting a new study on the roots of rice plants, they could study the arsenic levels and how the arsenic gets into the rice plants/roots (so we possibly may have a new research study underway in the next few months).
After the lab meeting, another member of the AGI team, Dario (who has been helping me a lot) and I ran a medium gel with the PCR products. I got to make the medium gel from scratch with about 1 g of agarose and 70 mL of TAE buffer (which is a common buffer used in gel electrophoresis and for those who are curious what TAE stands for -- it is Tris-acetate-EDTA). The results of the gel showed that one of the rice species (O. glaberrima) was highly concentrated so it did not clearly show the results of the other two rice species (O. barthii and the man-made heterozygous mix of the two species). So, I prepared another PCR but diluted the DNA. (I also accidentally diluted the O. barthii when I wasn't supposed to -- this just shows how important proper communication is in a lab -- but it was fine). I then continued to prepare a new PCR mix and loaded it into the PCR machine.
That was the end of my week! Hope you guys have another great week! I look forward to your next posts! See ya'll then!
On Monday, Feb. 17th, I started to run gels with my mentor Naomi. We ran a small gel with a primer called up2 which was a primer that is a little before the target segment of DNA that we're looking at. Later, we scored some older gels that the AGI team made and took pictures of and decided to re-run some of them. So we ran large gels with PCR plates that were made before. I got to insert the DNA using a 12 channel multi-channel pipette. At first, it got a little tricky trying to make sure I got all the PCR products inside each pipette and that I put all of the product inside each well and the pipette kind of gets unbalanced as you pipette out the PCR products. So I also did some practice pipetting with water and a plate.
 |
| 12 Channel Multi-Channel pipette |
 |
| Large gel with down1 primer (it might be kind of hard to see) |
On Wednesday, I observed my mentor preparing the PCR with the new primer solutions I made. After loading the PCR mixes into the PCR machine, I went into my second lab meeting. The more I see these lab meetings, the more I think, "Good thing we got lots of practice presenting during our capstone classes." Although these lab meetings are more of discussions and informal presentations, I could notice things that went well and things that didn't. But anyway, the main topic for this lab meeting was on new data relating to SNPs and imputations. I'm not sure what those terms mean and I think the other members of the AGI team didn't quite understand either, so a lot of time was spent trying to understand how the data was computed. At the end of the meeting, there was a discussion about rice and arsenic (which thankfully I had some background info in). It seemed as though there was more arsenic in the food supply in general than what everybody thought. Although there were jokes about how this fact might negatively affect the rice project the AGI team was doing (since they said rice was the answer to the 9-billion people question and could feed the world). However, it also seemed like the arsenic was coming into the food supply not only from natural causes such as volcano eruptions but also from human activities such as using pesticides/herbicides, mining, and burning coal and oil. So there were also discussions of how this issue of rice and arsenic can also provide a new path for research since the team was already interested in starting a new study on the roots of rice plants, they could study the arsenic levels and how the arsenic gets into the rice plants/roots (so we possibly may have a new research study underway in the next few months).
After the lab meeting, another member of the AGI team, Dario (who has been helping me a lot) and I ran a medium gel with the PCR products. I got to make the medium gel from scratch with about 1 g of agarose and 70 mL of TAE buffer (which is a common buffer used in gel electrophoresis and for those who are curious what TAE stands for -- it is Tris-acetate-EDTA). The results of the gel showed that one of the rice species (O. glaberrima) was highly concentrated so it did not clearly show the results of the other two rice species (O. barthii and the man-made heterozygous mix of the two species). So, I prepared another PCR but diluted the DNA. (I also accidentally diluted the O. barthii when I wasn't supposed to -- this just shows how important proper communication is in a lab -- but it was fine). I then continued to prepare a new PCR mix and loaded it into the PCR machine.
That was the end of my week! Hope you guys have another great week! I look forward to your next posts! See ya'll then!
Monday, February 17, 2014
Week 2
Hi guys. I hope you guys have had a great week at your internships.
On Friday, I was able to see another demonstration of PCR and making a gel. I am also getting myself more familiarized with the lab, where things are, and which doors to get in and out of (it gets confusing sometimes). I was also able to help prepare the PCR and it seems like I still need to practice my pipetting. But the process is basically the same as what we did in our capstone biology class.
I have also continued to do my research and my supervisor gave me a side note to look up arsenic poisoning in rice. Apparently the FDA found traces of inorganic arsenic (which is known as a human carcinogen) in rice since around 2012. They are in what the FDA considers very small amounts ranging from about 11 to about 160 parts per billion per serving in rice and rice products. The public might disagree with these being small amounts because on average, single servings of some rice products exceed the arsenic limit of drinking water which is 10 parts per billion (as determined by the EPA). Brown rice seems to have the most arsenic because it is not as refined or polished since the arsenic is usually clustered around the seed hull (which is the outer portion). Also, instant rice and rice wine tend to have the least arsenic. Although the FDA has analyzed arsenic levels in over 1300 types of rice and rice products, they are continuing their research since the levels tend to very greatly sample to sample, even within the same product. However, thus far, the FDA has concluded that the arsenic levels in rice will not have any immediate or short term adverse health effects, and they do not advise changing the people's consumption of rice. Ironically, they also advise to have a more balanced diet and also consume other grains and not daily eat rice.
With the rest of my research, I have been looking more at the history of rice and its domestication. The history of rice gets pretty complicated as it seems like it has been twice domesticated and twice de-domesticated. There are also many species of rice, the most common being Oryza sativa and Oryza glaberrima, and there are many subspecies such as under O. sativa are known as japonica and indica. The first is typically found in Asia and the second in West Africa. These two are 2 domesticated species of rice and are believed to have come from wild species called O. rufipogon and O. barthii, respectively. After some species of rice were domesticated, it seems that there was a weed that resembled an Asian rice variety and another strain that resembled rice grown in the tropics. This made and continues to make scientists question, did the domesticated rice revert back to their wild forms or were there mutations?
I have also been researching phenotypic traits besides shattering that differs between wild type and domesticated rice and also began researching the genotypic relationships. For example, wild types have very long awns while domesticated rice has short awns if any. Also, wild rice has higher dormancy levels while domesticated rice has reduced dormancy for uniform germination (making it easier to harvest all the rice grains at one time). Also, the pericarp and seed coat of wild type is typically red while domesticated rice is white, and grain size are also small in wild type and domesticated rice has varied sized grains. The panicle structure in wild rice is an open panicle with few secondary branches that carry only a few grains while in domesticated rice is a densely packed panicle that carries a large number of seeds. I am still trying to wrap my head around the parts of a rice plant and trying to understand all the data related to the genotypic information.
I will be looking more closely at the shattering genes and researching the certain genes and hoping to understand all the science language in these journals. Until then, hope you guys have a great week.
This week I have finally started my internship and figured out a schedule. I still need to work on my training which officially won't be over until the 25th where I have to take a course. However, I can work in the lab! (under supervision, of course)
On Wednesday, I was able to go to my first lab meeting. Unfortunately, the guy who was supposed to lead the meeting couldn't make it that day, so I didn't get to hear a whole bunch of science-y terms or hear about the new data for the rice genome project. On the other hand, I was able to listen in on a program called "R" (yes, it is only one letter). I'm still trying to wrap my head around it, but what I got was that it is a language programming system that can be used for bioinformatics and statistics and so on. What I got from the demonstrations of using R so far was that it was almost like a huge computer-version of a graphing calculator. I believe I'll get to see more demonstrations and hear more about it at the next lab meeting. I think I'll ask if it can make 3D graphs (but we really don't need any of that in biology). But it involves like vectors and components of vectors, assigning characters to values, and all that stuff. Oh, and the good thing is...it's FREE!
I also got a pipetting 101 lesson. I had to test the accuracy of 3 different pipetts by pipetting back and forth water. Although it was super tedious, not only did I see that the pipettes I used were accurate, but I was also complemented for being a good pipett-er. I think the purpose of the exercise was to pay attention to accuracy and practice my pipetting to make sure I get it right every time.
I have also continued to do my research and my supervisor gave me a side note to look up arsenic poisoning in rice. Apparently the FDA found traces of inorganic arsenic (which is known as a human carcinogen) in rice since around 2012. They are in what the FDA considers very small amounts ranging from about 11 to about 160 parts per billion per serving in rice and rice products. The public might disagree with these being small amounts because on average, single servings of some rice products exceed the arsenic limit of drinking water which is 10 parts per billion (as determined by the EPA). Brown rice seems to have the most arsenic because it is not as refined or polished since the arsenic is usually clustered around the seed hull (which is the outer portion). Also, instant rice and rice wine tend to have the least arsenic. Although the FDA has analyzed arsenic levels in over 1300 types of rice and rice products, they are continuing their research since the levels tend to very greatly sample to sample, even within the same product. However, thus far, the FDA has concluded that the arsenic levels in rice will not have any immediate or short term adverse health effects, and they do not advise changing the people's consumption of rice. Ironically, they also advise to have a more balanced diet and also consume other grains and not daily eat rice.
With the rest of my research, I have been looking more at the history of rice and its domestication. The history of rice gets pretty complicated as it seems like it has been twice domesticated and twice de-domesticated. There are also many species of rice, the most common being Oryza sativa and Oryza glaberrima, and there are many subspecies such as under O. sativa are known as japonica and indica. The first is typically found in Asia and the second in West Africa. These two are 2 domesticated species of rice and are believed to have come from wild species called O. rufipogon and O. barthii, respectively. After some species of rice were domesticated, it seems that there was a weed that resembled an Asian rice variety and another strain that resembled rice grown in the tropics. This made and continues to make scientists question, did the domesticated rice revert back to their wild forms or were there mutations?
 |
| Wild type v. domesticated rice phenotypes: (A) Immature panicle from O. rufipogon; (B) Mature panicle from O. rufipogon with dark hulls and long awns; (C, F) Dehulled seed from O.rufipogon (C) and O. sativa (F); (D, E) Grain bearing O.sativa ssp. japonica (D) and ssp. indica (E) panicles w/ straw-colored hulls w/ closed structure. (http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2759204/figure/MCM128F1/) |
I have also been researching phenotypic traits besides shattering that differs between wild type and domesticated rice and also began researching the genotypic relationships. For example, wild types have very long awns while domesticated rice has short awns if any. Also, wild rice has higher dormancy levels while domesticated rice has reduced dormancy for uniform germination (making it easier to harvest all the rice grains at one time). Also, the pericarp and seed coat of wild type is typically red while domesticated rice is white, and grain size are also small in wild type and domesticated rice has varied sized grains. The panicle structure in wild rice is an open panicle with few secondary branches that carry only a few grains while in domesticated rice is a densely packed panicle that carries a large number of seeds. I am still trying to wrap my head around the parts of a rice plant and trying to understand all the data related to the genotypic information.
I will be looking more closely at the shattering genes and researching the certain genes and hoping to understand all the science language in these journals. Until then, hope you guys have a great week.
Saturday, February 8, 2014
Week 1
Hey guys.
I should probably first talk about what I am going to be doing with this Senior Research Project. I am going to be interning at the University of Arizona Genomics Lab under my supervisor Dave Kudrna and my mentor Naomi Rhodes, who is an undergraduate at the U of A, and I will working on plant genomes, specifically, on rice from the genus Oryza. I will be focusing on the shattering trait of these genomes.
Unfortunately, I haven't been able to do much at the laboratory because there have been issues with my lab safety training. I finished half of it, which was the Laboratory Chemical Safety Training, but there have been problems with the other half which is the biosafety training. I hope to resolve the issue by early next week (Monday or Tuesday) and start working in the laboratory. After resolving the issue and finishing my training, I can have a set schedule for my internship.
On the other hand, I got my own little office space. I will be doing some of my research, reading, and writing there (and at home ^-^). I also did some reading on my topic, and as I was reading, some of the papers and articles answer questions like "Why are we studying this?" and "Why should we care?"
This project with the Oryza rice genomes hopes to answer part of the "9 billion-people" question. According to the article "The 9 billion-people question" in The Economist, it is estimated that by 2050, our world population will have increased from almost 7 billion to 9 billion people. Sooo...how will we feed all these people? When we look at our food supply compared to even today's population, we can barely feed everybody (and the food prices are not helping). This crisis brings up Malthusian fears and if we keep this up then how are we going to feed 9 billion people in 40 years? Well, rice, especially Asian rice (known as Oryza sativa), feeds more than any other crop in the world, and the rice-dependent population is expected to at least double in the next 25 years. Therefore, scientists must find a way to produce/grow twice as much rice as efficiently as possible. So, how is shattering related to this? Well, first, you might ask, "what is shattering?" (I mean, I kept using the word, I should at least define it or give some background, right?) "Seed shattering is an adaptive trait for seed dispersal in wild plants...[and it] causes yield loss for domesticated crop plants during harvest." Our ancestors began domesticating rice when they started selecting rice with less shattering (Zhou et al. 1). Reducing shattering in wild rice species may be a way to help domesticate rice making it easier to harvest. So we also get into some evolution and history of Oryza rice plants.
Although I haven't been able to do all the fun lab stuff yet, I am really looking forward to doing experiments and learning about the thing that I eat most at home (RICE!).
I hope you all have a great weekend and for my fellow seniors, another fantastic week at your internships. I will look forward to your postings. Until then, ta-ta for now.
I should probably first talk about what I am going to be doing with this Senior Research Project. I am going to be interning at the University of Arizona Genomics Lab under my supervisor Dave Kudrna and my mentor Naomi Rhodes, who is an undergraduate at the U of A, and I will working on plant genomes, specifically, on rice from the genus Oryza. I will be focusing on the shattering trait of these genomes.
Unfortunately, I haven't been able to do much at the laboratory because there have been issues with my lab safety training. I finished half of it, which was the Laboratory Chemical Safety Training, but there have been problems with the other half which is the biosafety training. I hope to resolve the issue by early next week (Monday or Tuesday) and start working in the laboratory. After resolving the issue and finishing my training, I can have a set schedule for my internship.
On the other hand, I got my own little office space. I will be doing some of my research, reading, and writing there (and at home ^-^). I also did some reading on my topic, and as I was reading, some of the papers and articles answer questions like "Why are we studying this?" and "Why should we care?"
This project with the Oryza rice genomes hopes to answer part of the "9 billion-people" question. According to the article "The 9 billion-people question" in The Economist, it is estimated that by 2050, our world population will have increased from almost 7 billion to 9 billion people. Sooo...how will we feed all these people? When we look at our food supply compared to even today's population, we can barely feed everybody (and the food prices are not helping). This crisis brings up Malthusian fears and if we keep this up then how are we going to feed 9 billion people in 40 years? Well, rice, especially Asian rice (known as Oryza sativa), feeds more than any other crop in the world, and the rice-dependent population is expected to at least double in the next 25 years. Therefore, scientists must find a way to produce/grow twice as much rice as efficiently as possible. So, how is shattering related to this? Well, first, you might ask, "what is shattering?" (I mean, I kept using the word, I should at least define it or give some background, right?) "Seed shattering is an adaptive trait for seed dispersal in wild plants...[and it] causes yield loss for domesticated crop plants during harvest." Our ancestors began domesticating rice when they started selecting rice with less shattering (Zhou et al. 1). Reducing shattering in wild rice species may be a way to help domesticate rice making it easier to harvest. So we also get into some evolution and history of Oryza rice plants.
Although I haven't been able to do all the fun lab stuff yet, I am really looking forward to doing experiments and learning about the thing that I eat most at home (RICE!).
I hope you all have a great weekend and for my fellow seniors, another fantastic week at your internships. I will look forward to your postings. Until then, ta-ta for now.
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