good party

thanks to connor, joe, and tyson for hosting an awesome party tonight. i ;m glad that both phil and miklos are back too. it's going to be a fantastic senior year with the two of them to hang out with. i can't wait.

still not that thrilled about classes starting soon, but glad to see everyone again.

and now it's time for some water and bed and sleeping.


ps. i heart gewit. but everyone already knows that.

world's best veggie dip

here's how you make it:

1 cup mayonnaise
1 cup sour cream
1 tbs dill weed
1 tbs beau monde
1 tbs parsley

you might be asking yourself, "and what exactly is beau monde?"

that's what i found out today:

beau monde spice mix

1 tbs ground cloves
1 1/4 tsp ground cinnamon
1 tbs salt
1 tbs ground bay leaf
1 tbs allspice
2 tbs ground black pepper
1 tsp ground nutmeg
1 tsp ground mace
1 tsp celery seed
2 tbs ground white pepper

mmmm... delicious with carrots!!

new links

really they're the same, just with different names. i didn't even change the order or anything.

woohoo it's friday and that means cola-beer floats in wizzo 403!

fluffies say hello.

life is interesting

so today wasn't the greatest day of all time.

but that's ok.

i am, however, worried about the possibility--or really the lack thereof-- of me taing the biology genetics lab this semester. my class schedule leaves me free to ta the monday/friday section of the class, but they are having trouble filling the positions for the tuesday/thursday section. the emails that i got today from the professor who teaches the class make me believe that i will have to choose between changing my class schedule to be able to ta the t/r section and keeping my schedule and not taing. that sucks. big time. i'm meeting with the professor tomorrow and i guess i'll figure stuff out then. but right now, i;m kind of disappointed and scared and sad that i won't be able to ta at all. life is poop and full of disappointments.

in other news, i am trying to pick a song to sing for auditions. i have it narrowed down to two: 1. "once you lose your heart" from me and my girl and 2. "happy to keep his dinner warm" from how to succeed. if you know the songs and have an opinion on which would be better, please post a comment and let me know.

i also had fun tonight hanging out with phil and josh einstein, even though gerrit could not get into hemmingways because they suck ( :( ). poop.

leave me comments because i like them.

my schedule

why not?

this is kinda crazy but oh well. take a
look.

new jersey

is where i am now.

my mommy missed me too much and made me come home for five days. so here i am.

here's what has happened recently (in no particular order):

my friend from hs, jeff, came to visit me and that was fun.
a trip to the goodwill resulted in new pants and a neato kitchen tool to make calzones and other pocket-type foods.
i learned a lot of new words that i forgot yesterday around noon.
i took the gres and got a 1420 (or it might have been 1410, i can't remember. either way, i'm happy).
i helped gerrit move into morewood by pushing his chair down fifth ave and looking like a fool.
everyone is starting to move back in for school and i am in new jersey.
i saw me and my girl starring jimmy brenan, this guy i met while a part of skit's production of crazy for you.
i'm thinking about auditioning for bat boy, possibly with a song from me and my girl.
i borrowed a million cds from the carnegie library of pittsburgh and hunt-- most of them musical soundtracks-- and copied them all.
i read in the park for almost two hours.
neil gaiman's stardust will probably be ranking among my favorite books from now on.
i went to the strip district and got bread and sunflowers.
saw modest mouse in a sucky concert.
watched lots of the olympics.

and some other stuff too. i can't remember everything.

still so much to accomplish this summer. i'm working on the sns scrapbooks, which is insane. i need to narrow down my list of grad schools to apply to. although first i need to make a list. so far the only school on the list is yale because they keep sending me stuff and it looks pretty.

i'm really happy to see people very excited about what they are doing this year. sort of a vicarious enjoyment on my part i suppose, but it's nice to see others having so much fun.

eh, i think that's enough.
nighty night.

haven't done any silly quizzes in a while....

stolen from ashley's blog:


Category III - The Regular
Jo(e)

You are the quintessential standard conjured by the
word 'Friend'.


What Type of Social Entity are You?
brought to you by Quizilla


i'm sad because my mommy is making me go home for a week. at home i will sit on the couch by myself because no one else will be home. there will be nothing to do because i live out in the middle of no place. at least i'll have lots of time to work on the sns scrapbooks....

still working on those gre words. i know lots more words now, but probably not as many as i should.

my RNA gel worked!!

hooray! my last day of work before the symposium and my rna gel finally decides to work. here's a picture:



the lane all the way to the right contains the control wild type rna. the top band is the 25s rRNA and the bottom one is the 18s rRNA. unfortunately, my samples are all faint, and it's hard to tell if there's any real difference in the levels of 25s or 18s. but that's ok because it worked!!!

fluffies!!

ps.

i welcome comments and questions.

now you can link to the tables and look at them too! how exciting!!

in other news-- completely unrelated to biology and ribosomes and work-- i bought myself a cute little dvd player this past weekend. it's so wonderful!

also, gerrit's fluffy has come to visit the rest of the fluffies. i don't have a picture, otherwise i'd post one. his name is arfie because gerrit thought he was a dog. how silly. but arfie likes hanging out with the rest of the fluffies too!

hooray for fluffies!!

on boredom, ribosomes, and dominant-negative mutants

what follows is the report that i am giving on my research this summer at the lab meeting tomorrow morning. it goes in order so you can just scroll down and read it in the correct order.

i'm really bored.

here's a diagram of the current view of ribosome synthesis for your enjoyment:


woohoo!

Ts-/Cs- Mutants

Tiffany had been trying to insert the YTM1 gene into pRS315 so that I could use the plasmid in my screens for Ts- and Cs- mutants. She had also been trying to insert ytm1-1 into pSLS315 for a future synthetic lethal screen. I mini prepped the transformants that resulted from her ligation reactions using the BioRad kit. Digests of both plasmids showed that neither ligation was successful. Because we were having so much trouble with the pRS315 ligation reaction, I decided to focus on finding dominant mutants of Ytm1p.

Dominant-Negative Mutants

In order to screen for dominant-negative mutants I used pBM258T/YTM1, which contains YTM1 inserted at the BamHI site. This puts the transcription of the gene under the control of the inducible GAL1 promoter so I can easily turn on or off the transcription and translation of the mutant alleles of Ytm1p.

PCR Mutagenesis:

PCR mutagenesis was used to create single point mutations in the YTM1 gene. I first tested my primers by doing a PCR reaction with the normal reaction conditions (200uM dNTPs, 600nM primers, 1ul of a 1:10 dilution of mini prepped template plasmid DNA, 1x buffer, and 1ul Taq enzyme). Analysis on an agarose gel showed that the PCR worked fine.

1. The first PCR mutagenesis reactions contained 10-fold molar less (20uM vs. 200uM) of one of the four dNTPs. Each of the four possible reactions was run along with a control containing equal molar concentrations of all four dNTPs (200uM). The other reactions conditions were standard. I repeated my PCR cycle 18 times.

2. The second PCR mutagenesis reaction contained the same reactions conditions and the same PCR program. This time I repeated the PCR cycle 20 times in hopes of getting a larger yield of mutagenized insert. However, a larger number of PCR cycles increases the possibility of having multiple mutations within each copied insert.

3. The third PCR mutagenesis contained template DNA (the plasmid pBM258T/YTM1) that had been washed with PCI and EtOH precipitated. The rest of the reaction conditions were the same as the conditions for the first two PCR mutagenesis reactions. I also used a longer elongation time—3 minutes vs. only 1 minute for the first two PCR reactions. Analysis on an agarose gel showed that more product was present with the longer elongation time, especially for the reaction containing 20uM dCTP.

Dominant-Negative Mutant Screen:

A total of 160 transformants—60 from the second gap repair transformation and 100 from the third gap repair transformation—were screened for inhibited growth on galactose containing media. The transformants were patched onto C-ura media and then replica plated:
1C-ura,
2C-ura+2%gal
3C-ura+4%gal
4complete.

In order to dilute out any glucose that may have remained after the initial replica plating, the C-ura+2%gal plates were replicated once more:
1C-ura,
2C-ura+2%gal
3C-ura+2%gal
4C-ura+4%gal
5complete.

After the initial replica plating five transformants showed no growth on the galactose containing media and two transformants displayed growth that was significantly slower than the control (transformant with the unmutagenized plasmid pBM258T/YTM1). All of these transformants came from the third gap repair transformation. After the second round of replica plating, additional slow-growing mutants were seen: one grew significantly slower than the control and four grew slightly slower than the control. There was no difference in rate of growth of any of the transformants on media containing 2% galactose or 4% galactose. These eleven mutants were chosen to be further studied.

Gap Repair:

I used gap repair to insert the mutagenized copies of ytm1 into the pBM258T/YTM1 plasmid. Because little is known about pBM258T, I had to test various restriction enzymes to find some that create a gap in the YTM1 insert. I tested the following enzymes that I knew cut in the YTM1 insert to see if they cut within the vector as well: SnaBI, AatII, SwaI, BsrGI, MluI, and XmnI. The only restriction enzyme that we have that cuts only in the insert and not the rest of the plasmid was SnaBI. PBM258T/YTM1 was digested with SnaBI to create linearized DNA (the enzyme only cuts once) and treated with alkaline phosphatase. This “gapped” plasmid was used in gap repair in yeast strain JWY3400 (ura-).

Three gap repair transformations were performed:

1. The first gap repair transformation used inserts created in the first PCR mutagenesis reaction. This transformation yielded zero transformants. Since there were a few transformants on the plate from the gap repair with the unmutagenized insert, I thought that the concentration of the mutagenized insert might have been too low for any results. I decided to repeat the transformation using 10ul of the various inserts instead of 5ul.

2. For the second gap repair transformation I used either 5ul or 10ul of each of the mutagenized inserts from both the first and second PCR mutagenesis reactions. Transformants were seen on most of the plates. There was no correlation between the volume of insert used in each transformation and the number of transformants. The number of transformants on each plate ranged from zero to approximately 300.

3. The third gap repair transformation used the inserts from the third PCR mutagenesis reaction that had been washed with PCI and EtOH precipitated. Transformants were seen on these plates as well. Again, the number of transformants on each plate ranged from zero to approximately 300. I did see the largest number of transformants resulting from the PCR reaction with the 20uM dCTP as compared to the other dNTPs.

Serial Dilutions:

In order to clearly show the growth phenotypes of these mutants on galactose containing media, serial dilutions were carried out. The first round of serial dilutions was not quantitated and the amount of cells in each sample was not consistent. This first round of serial dilutions included all eleven putative mutants. Dilutions of 10-1, 10-2, 10-3, 10-4, and 10-5 were plated onto C-ura, C-ura+2%gal, and C-ura+4%gal and incubated at 30 deg C for 5 days. The expected phenotypes were seen for four of the mutants that did not grow on galactose and the three mutants that grew significantly slower than the control. The other four mutants did not display significant differences in growth when compared to the control. One of the mutants that did not grow on galactose turned out to be contaminated with mold.

The serial dilutions were repeated because I wanted to have the same concentration of cells in each sample and because mold grew on some of the plates. I only studied the seven best mutants. This time, after inoculating one colony of each putative mutant I took the OD610 to determine the concentration of cells and diluted the samples so that the number of cells in each spot would be the same for all the samples. Dilutions of 1, 10-1, 10-2, 10-3, and 10-4 were plated onto C-ura and C-ura+2%gal and incubated at 30 deg C for 5 days. Again the expected phenotypes were seen: four mutants did not grow at all on the galactose media and three grew more slowly than the controls. These serial dilutions on C-ura and C-ura+gal are shown in figure 1.

Figure 1: Serial Dilutions of Putative ytm1 Mutants Showing Lack of Growth or Slow Growth on Galactose Media

RNA Extractions:

In order to further characterize the dominant-negative mutants I had collected, I decided to screen for defects in the synthesis of rRNAs by using RNA extraction to find the relative levels of rRNA in the cells. I extracted the RNA from the four mutants that did not grow at all on galactose, the three mutants that grew slowly on galactose, the mutant that was contaminated with mold, the control containing the original pBM258T/YTM1 plasmid, and two controls containing a gap repaired pBM258T/YTM1 with an unmutagenized insert (one from each of transformations 2 and 3).

1. For the first RNA extraction, I forgot to shift the cells from glucose media to galactose. Each sample was resuspended in 20ul dH2O. This extraction served as a practice. I ran my samples on a 1% agarose gel (not very RNase free) to get an idea of how much of each sample to load on a gel in the future. 20ul of each sample seemed to be more than enough; 10ul would have also worked. Because my gel was not RNase free, the bands came out as very big smears—the RNA had been degraded.

2. For the second RNA extraction, I grew up my cells in 5ml of C-ura overnight and then shifted them to C-ura+gal for another overnight. For controls, I grew up two cultures of JWY3400 (the parent strain) in YEPD and shifted one to YEPGal. I inoculated the other culture into YEPD. I harvested the cells during log phase growth (OD = ~0.4), extracted the RNA, and resuspended my final pellets in 20ul dH2O. I ran 20ul of each sample on a 1% agarose gel. However, as I was loading the gel, my samples started leaking out of the wells. I ran the gel and found that most of the sample had left the wells. The only RNA was located in the wells still; nothing had run down the gel.

I also took the OD260 of each sample to find out how much RNA was present in each sample. I diluted 2ul of each sample into 500ul dH2O for a 1:250 dilution. The levels of RNA present in each sample varied greatly. Table 1 shows the concentrations of RNA in each sample.

3. When I repeated the RNA extraction the third time, I made all new solutions in order to ensure that everything was RNase free. I grew up the cells in 5ml C-ura overnight and shifted has I had during the second RNA extraction. I used the same controls as well. I extracted the RNA and resuspended in 20ul. This time I ran only 10ul of each sample on a 1% agarose gel. When I loaded the gels, I had no problems with dye leaking out of the wells as I had seen before. However, after I started running the gel, I noticed that some of the dye had started leaking out. I let it run for 15 minutes and then checked to be sure that some of the RNA sample had traveled into the gel. Once I saw RNA in the gel, I let it run for 2 more hours at 80V.

The RNA gel shows that most of the RNA in the wells had floated out, as shown in figure 2. The RNA that I had seen in the gel did not move in the two hours that the gel was running.

Again, I took the OD260 of a 1:250 dilution of each of the samples. As before, I could see no clear correlation between the level of RNA in the sample and the growth rate of the cells on galactose. The levels of RNA varied greatly throughout the samples. Table 1 shows the RNA concentrations of the samples.

Figure 2: 1% Agarose Gel of RNA Extractions From Putative ytm1 Mutants

Verification of Mutant Phenotype:

To verify that the mutant phenotype was cause by the plasmid in the cells, I shuttled the putative mutant plasmids from yeast to E. coli and then mini prepped the plasmids. I shuttled the plasmids from the four mutants that did not grow on galactose, the three that grew at a much slower rate than the controls, the mutant that was contaminated with mold, four mutants that grew only slightly slower than the controls, the control with the pBM258T/YTM1, and three controls containing the gap repaired pBM258T/YTM1 with each of the three unmutagenized inserts from the three PCR reactions. The transformation of the extracted yeast DNA to DH5-a cells was done with electroporation and yielded between 100 and over 1000 transformants for each sample. After mini prepping one transformant of each sample with the BioRad Kit, I transformed the plasmids back into yeast cells (JWY3400). I also set aside some of the mini prepped plasmids for sequencing.

The transformation to JWY3400 yielded more colonies than I could count for all but two of the transformations. One of these lower yield plasmids was a mutant that only grew slightly slower on galactose than the control and the other was a mutant that did not grow on galactose. These transformations still yielded over 50 transformants each. I patched three colonies from each of the transformation plates onto C-ura, grew the patches overnight, and replica plated:
1C-ura
2C-ura+2%gal
3C-ura+4%gal
4complete

After incubation at 30 deg C for two days, growth could be seen in all the patches, including those on the galactose media. In order to dilute out any glucose that may have still been present in the patches, I replica plated a second time:
1 C-ura
2C-ura+2%gal
3C-ura+2%gal
4complete

Again, I saw even growth in all the patches on all the plates except for the negative control (JWY3400). I decided to patch three more colonies from each transformation plate and replica plate again:
1C-ura
2C-ura+2%gal
3C-ura+2%gal
4C-ura+4%gal
5complete

I am still waiting to see if there is growth in these replica plated patches.

Site Directed Mutagenesis:

In order to be able to create a larger gap in the YTM1 gene in the plasmid pBM258T/YTM1, I attempted to create a second SnaBI restriction site by site directed mutagenesis. I talked to both Carol and Jelena about their previous site directed mutagenesis experiments, and modeled my reaction conditions after them. My primers were 39 nucleotides in length and had a Tm of 79 deg C—good conditions for creating two mutations separated by only four bases. I tried many different PCR reaction conditions, which are outlined in table 2. For all of the various reactions, I followed the basic procedure outlined in the Stratagene kit protocol. I mutated my plasmid using PCR, digested the parent plasmid with DpnI and transformed into competent DH5-a cells using electroporation. Because I was working with a large plasmid (9.6kb) I used the high fidelity Pfu Ultra DNA polymerase for all of the reactions.

1. The first set of site directed mutagenesis reactions yielded no transformants. This may have been due to the age of the competent DH5-a cells used. Also, I did not purify the DNA in the reactions after digestion with DpnI.

2. For the second set of reactions I purified the DNA after digestion with DpnI by precipitating and washing with ethanol. The ethanol purification helps the transformation because it gets rid of salts that are in the final site directed mutagenesis solution with the DNA. Electroporation does not work as well when salts are present. I did two different transformations: the first transformation was into the older competent cells and yielded only one transformant. However, when I repeated the transformation with new competent cells, I got thousands of colonies.

3. Again, I had purified the DNA by precipitating and washing with ethanol for the third set of reactions. I also used the new competent cells. Although I saw a large number of transformants on one of my plates, I also saw an even greater number of colonies growing on the negative control that contained no DNA. I need to repeat this transformation.

I began screening through the transformants. If two SnaBI sites are present in the plasmid, I expect to see two bands when I run the digested plasmids on an agarose gel: 8759bp and 881bp.

1. The first transformation of the second set of reactions had yielded one transformant. I mini prepped the plasmid with the BioRad kit and digested with SnaBI. Analysis of the digest on a 1% agarose gel showed only one band at the same size as the control pBM258T/YTM1 digest (9640bp). A second SnaBI site had not been created.

2. I picked 12 transformants from the second transformation of the second set of reactions and mini prepped using the regular protocol and not the kit. I digested with SnabI and ran the digests on a 1% agarose gel. I saw only one band at about 9640bp for each of the digests. This is shown in figure 3. I had trouble interpreting the agarose gels at first because there was a lot of degraded RNA present in the bottom of the gel. However, it was pretty clear that only one band was present. No bands were seen around 900bp. Again there was no second SnaBI site formed. I picked another 24 transformants, mini prepped with the regular protocol, digested with SnaBI, and ran the digests on a 1% agarose gel. As before, I only saw one band. In order to make sure that I was seeing only one band on the gel, I chose six of the transformants and mini prepped them with the BioRad kit. I digested with SnaBI and ran the digests on a gel. It was very clear this time that there was only one band at 9640kb. This is shown in figure 4.

I am continuing to screen through transformants from the second set of site directed mutagenesis reactions.

Figure 3: Example of Site Directed Mutagenesis Results- Regular Mini Prep Protocol


Figure 4: Site Directed Mutagenesis Results- BioRad Mini Prep Kit