So much to do, so little time.

Hello VDSers!!! I hope you all had a great and restful break with good food and company. I CANNOT believe that it is the final week of class. I don’t know where the time went and I feel like this has been my toughest and fastest semester yet. But, it’s only going to get tougher and we all have to finish strong!

Before the lovely break, I worked on the actual pNIC-Bsa4 cloning. I got a successful PCR to work the last time from my original PCR and I chose Sample C (As mentioned in my bog post on 11/22/2010). So, since I prepared pNIC-Bsa4 as an accepting vector the day before I started cloning, I continued on topreparing the “Cohesive End Generations” for both my Accepting Vector and my PCR inserts.

For the PCR Insert I mixed in a 1.7 tube:

12ul of my PCR fragment (Sample C), 2ul of T4 DNA Polymerase Buffer(Buffer2) , 0.5ul 10X mM dCTP, 1ul of 100 DTT, 0.2ul of 100XBSA, 1ul T4 DNA Polymerase, and the rest was H2O.

For my Accepting Vector mixed in a 1.7 tube:

When I performed the PCR cleanup on my preparation of pNIC-Bsa4 as an Accepting Vector, in the final step it asks you to add 50ul of Elution Solution. Well, I accidentally added 500ul, which meant that it was diluted by a lot. So, this forced me to change the original amount of the plasmid and H2O.

39.5ul of half of cut plasmid, 5ul of 10X T4 DNA Polymerase Buffer (Buffer 2), 1.25ul of 100mM dGTP, 0.5ul 100X BSA.  1.25ul T4 DNA Polymerase, NO H2O.

These were both left at room temperature for 30 minutes (~22C) and were then heat inactivated at 75C for 20 minutes. The step that was supposed to be followed immediately after this was the annealing and transforming step, but was followed an hour after instead due to a mandatory meeting. My samples were left in the 4C while I was at my meeting.

For the annealing and tranformation step:

I made two different tubes (A & B). Tube A consisted of 2ul of T-4 treated Accepting Vector along with 4ul of each T-4 treated Inset. Tube B consisted of my own mixture and I added 2ul of the Accepting Vector and 8ul of the Insert.

They were left at room temperature for 10 minutes, moved to ice, and then 25ul of competent cells were added. They were then left on ice for another 30 minutes, heat shocked for 30seconds at 42C , put back on ice, SOC was addedand they were then left on the 37C shaker for an hour. I did not have KAN+Sucrose plates available for me, so Dr. Beckham allowed me to used KAN plates alone. I spread all of the itranformationl mixture to the plate using colirollers and they sat in the 37C overnight to grow.

BUT, you all know how sensitive this stuff is, and it FAILED 😦 This is a picture of my plates that I took. As you can see…they have nothing grown in them!

Today, I am doing this again in hopes that I can at least have something work by Friday or Saturday. Good luck with to all of you all in lab and with your studies. The year is almost over and we have to enter 2011 with a good attitude and smile!

Long Weekend

Hello all you lovely VDSers. I hope you all had a weekend.

Last week I FINALLY got my PCR to work!!! Holy cannoli, am I behind. So, this week I worked on my scaled-up PCR and I started the actual protocol for pNIC-Bsa4 Cloning. On Friday, I followed the “Protocol of PCR for pNIC-Bsa4 Cloning”, but I multiplied all the volumes by two. I chose my ‘Sample C’ from my first PCR to scale-up. That Sample C consisted of14.25ul of the ‘Master Mix’ which had (17.5ul 10x- KOD Buffer #1, 26.25ul dNTP, 28ul of both FOR and REV primers), 2ul of MgCl2, 6.5ul of H2O, and 1.25 ul of DNA Template).  So, I basically just doubled all of those amounts and made Samples A-F of Sample C.

The first one of that came out like this: (Scaled-up PCR of Sample C)

Yes I know. It’s really bright. I realized later that I keep forgetting to dilute the KOD DNA Polymerase. If you look at Lane 10, it should be empty, but it isn’t. That’s because some of Sample F (of Sample C) spilled over. I didn’t think too much about that until Saturday when I came in and asked Leighana what she thought. She told me that I was supposed to save 1/2 of my PCR samples for PCR Cleanup 😦 I always forget to do things. Speaking of which, I forgot to not add the DNA template to Sample F. I know, I’m ridiculous.

I also worked ion preparing pNIC-Bsa4 as an accepting vector, as previously mentioned. I added used pNIC-Bsa4 plasmid that had an initial concentration of 228ng/ul. I only needed 2.25ng of that, so that came out to needing only 9.86ul of the pNIC-Bsa4 plasmid along with2.5ul of Buffer3, 0.25ul of BSA, 1ul of BSAI, and 11.39ul of Nanopure H20. I left that at 50C for 3 hours, then did PCR cleanup on it hping to work on it on Saturday.

So, on Saturday, I did another PCR with diluted KOD and I ONLY used 25ul out of the 50ul samples. Everything went well except for the fact that I received some news from home and decided to head back to my apartment. Thanks Leighana for being there. This is a picture of my PCR from Saturday (Finally done right!!!)

So that was that.

Today, I was so READY to keep working on my cloning. I was going to begin the ‘Cohesive End Generation’ steps for both my PCR and my Accepting Vector. I got everything ready and then realized that I have no Sucrose+KAN plates. Even if Ihad wanted to do it yesterday, there was no mentor to do it with. So, hopefully Dr. Beckham can help me with that.

As of now, I have my preparation done for pNIC-Bsa4 as an Accepting Vector, and I am ready to move on as soon as I have a mentor. For now, I have a presentation to work on for tomorrow! I hope I can keep you guys awake!

I’m Back!

Hey VDSers,

I hope you all had a good weekend. I just finished my 5th PCR. Is that even normal??? How many PCRs did you all have to do? Anyway, this is a summary of what I did since I last posted:

I really wanted to try and do this on my own, so I did and failed AGAIN, miserably.

 

So, yeah. That sucked. Okay- So, I finally asked Radhika what she did when she got a successful PCR. I followed her steps, but I suppose the DNA template I used was wrong because I had bad results.Then, on Friday, I got these results:

So, Dr. Beckham had me use another DNA template, but run with Radhika’s program anyway. The DNA template that I used belonged to Candace and had a concentration of 560.6 ng/ul. I made a 1:100 dilution and it came out to a 7.2 ng/ul concentration. That was good enough for me. I used 1.25ul in each of my sample tubes. These were my results:

I’ve never seen anything like this. So, can any of you all help? I’m not really sure what this means. I do know that it didn’t work, but I’m not sure. I need Dr. Beckham!

So, yes, those were my last few weeks. Failing PCRs. I just want to finish this cloning and move on to the fun stuff again! I’m so jealous of you guys!!! Anyway, I still have yet to finish my PyMOL refresher, so I’m on that. Have a good night guys. See you in lab tomorrow!

PEOPLE PEOPLE PEOPLE!

Sorry I haven’t been posting. Busy busy busy. I will post sometime this week with a summary of the last two weeks. See you in class!

Not again!!!

Hello everyone. I hope this blog finds you well. I just finished running my gel on my PCR for the third time. And, I think I need to talk to Dr. Beckham about what I am doing wrong. So, here is the image:So, as you can see, I don’t think I got any good results. I did the same thing I did last ween using the PCR for PNIC-Bsa4 cloning protocol, but I got nothing. So, I need help!!! You’ll probably see me in lab this week since I need a mentor, or Dr. Beckham.

Well, I’m off. Have a good Sunday, everybody!

PyMOL Image Contest

I was feeling a little fruity. I hope you all like it!

It looks better when it’s bigger. Anyway, this is PTP1b. The Oxygens are shown in yellow, the Nitrogens are shown in light blue, and the Carbons are shown in red/pinkish. The active site of PTP1b is shown in the aqua green/sea foam green and the ligand that I tested in September (5753084) is shown in purple. Although this ligand did not inhibit PTP1b’s activity, I still put it.

PCR just doesn’t like me.

HELLO VDSersss! I hope everyone had a great weekend! I just ran a gel on my PCR and it failed…AGAIN! I am beginning to finally feel everyone’s pain. But, I think I FINALLY know what happened. Well, I’m actually positive.

So, ALL of my bands are bright. And I know I did not have a successful run for all of them. So, I am positive that I added way too much primer. (I forgot to dilute it) 😦 I added that onto my notebook already so that I do not forget!!! Also, I ran this at a temperature of 61.3, which I suppose is way too high. I just checked the idt website again, and now I think I finally got it right since I found a temperature that is lower that 55. So, I hope that it works next time around because I am so behind!!! Oh yeah, I used a 1KB ladder because I couldn’t find a 100 bp one.

Well, that’s it folks. I hope you all have a good week. By the way, I usually do labs on Friday’s and weekends since I’m working 15hrs at my job, so if you all ever need to come in but don’t want to be alone…I’m here 🙂

NOOOOOOOOOOOOOOOOOOOO!

Son of a hoarder! Nooo! My PCR was a complete (beep, beep, beep) FAIL!!! 😦

Well, I hope this blog finds everyone well! This week, I got sent back to working on PNIC-Bsa4! My glory days are over! Well, I need to go through the process, so it’s okay.What can I say, I really like the lab.

Anyway, so this week I worked on PCR for PNIC-Bsa4 cloning. My target is PTP1b, which, come to think of it…I’ve never talked about it on this blog. Well, I used the Forward and Reverse primers (VDS 13&14) for PTP1b, dNTP, 10X KOD Buffer, Water, KOD, and of course, my DNA template to run the PCR. I tried to be really careful with this PCR, but my results, as I previously mentioned, were not what I wanted them to be. So, I’m quitting VDS. DUN, DUN, DUN!

…Just kidding 😀 I’m just done for the day.

I think what happened was that I did not have the accurate annealing temperature. I will look into that for the next PCR this upcoming week. Here is a picture of my gel:

Yes, it stinks! I hope to get a successful PCR run next time I am in lab. Well, I hope everyone has a great and productive Saturday!

Back in Austin

Hello everyone. I hope this post finds you well. This week, I had the great opportunity to present the research that I have been working since the summer to many graduate students, researchers, scientists, and fellow peers. I had a blast. I didn’t think that I would have that much fun presenting my work and talking to people about what I am doing at the university. There were six other students from UT that traveled with me to El Paso along with Sarah Simmons. We all got to know each other really well.

It was pretty much a poster session, but it was a lot more intimate. I had over ten people come up to my poster and we talked about what I did. My throat was so sore after it was over. Because it was a competition, I did get judged. I did not place, but hey, that’s okay. Actually, no one from our institution placed. However, I really do think that we all did a great job. For most of us, it was the first time we had ever done something like that. That alone was rewarding. I did get my scores from the judges, and I did really well. It was based on a scale from a 1-4. For the most part, I got 4’s, but also saw a few 3’s. Anyway, that wasn’t what mattered to me. I gained a whole lot of experience through this, and realized that I like presenting. I met so many interesting, intelligent, and ambitious people in El Paso that had very intriguing projects.

Overall, it was a great conference. I feel very lucky for getting this opportunity. If you all ever need help with setting up your poster, or if you all have any questions about what to do, let me know! I think it would be smart to look at templates before hand because the spring is closer than you think and it sucks when you leave things to the last minute.

Now I am back inAustin and am NOT ready for the hell that is waiting for me these next two weeks. Seven pictures due for my photography class, two exams in one day, three assignments due, Texas 4000 Application due, lab report, and trying to fix the monster under my bed (my lab notebook)! Oh boy, I have a lot of work to do, as I’m sure we all do! See you all tomorrow!

Hectic Week

Hello VDSers! So, I know I’m late since I didn’t post last week and I’m a day late for this week. Well, I’m here now! I hope everyone is doing great, and not freaking out about the first exams of the school year (I know I am!!!).

Well, this past week has been a little hectic. I’m not stressing out because I know that everything can get done when you want it to, but I am concerned about how much time I don’t have! For those of you who don’t know, I am presenting my research in the LSAMP conference in El Paso in a few days. YIKES!!! I found out I got chosen for this great opportunity during the last few weeks of the summer and did not realize how soon September would arrive. I will leave on September 16th, and I will return on September 19th. So, that means that I need to yield results by tomorrow or Wednesday morning because I have to print out my poster by Wednesday before 5:00PM!!! So, that’s why you all have been seeing me so much in the lab this past week.

So, the week of 08.30.2010, I was trying to figure out where I had left off to continue the following week. I realized that I had made brand new protein, but failed to add glycerol, which basically means that what I made is no longer any good. Dr. Beckham had me express more protein last week, and for those of you that have not expressed protein, you will find that you need to devote pretty much an entire day to doing so. Luckily, I was able to devote all that time on Friday, which I should have devoted on Monday.

So, on Wednesday, I picked a single colony from my PTPib plate, and let it grow over night. I soon realized that I wouldn’t be able to work on it on Thursday because as I said, it takes an entire day! (I have class from 8AM-8PM on Tuesdays and Thursdays) So, basically, that was not any good, and so I did the same thing on Thursday (the next day) and let it grow over night in the 37C incubator. On Wednesday I also began running my real target on GOLD!!! It was really exciting. Dr. Beckham helped me out with that. On Friday, I came in bright and early to the lab, and checked my overnight cultures, and handled them appropriately. I did that all day, and was even able to fit in some enzyme assays later that evening from protein that was used in the past (A total of 8 samples). The readings that I got on those assays were the following (Sample 1: 0.44, Sample 2: 0.650, Sample 3: 0.113, Sample 4: 0.165, Sample 5: 0.173, Sample 6: -0.060, Sample 7: 0.082, Sample 8: 0.081) Only the first two samples (1&2) turned yellow (which is a good thing) and the rest did not. The last two (7&8) are the ones that I made in the summer and did not add glycerol to 😦 I’m still not too sure what these readings mean. But, I will in the next post! On Saturday, I came into the lab to sonicate and spin down my protein samples. That was successful. I also followed up on my GOLD run and found out it was finished. However, my GOLD scores did not look all that good, so I ran another library.

Today, I am purifying my enzyme, will run a gel, and will begin on inhibition assays with the protein samples that worked on Friday. Wish me luck everyone. I’m going to need it.