In the lab report instructions, it called for 2 figures of your own data (on pg 3), but only include gels that worked. I only have two gels (one that semi worked for Cycle Course PCR and one that worked for Cycle Course PCR). Can I include both of them in my lab report, because I already labeled them and I don't have any other gels?
Thanks
Undergraduate Research Forum
The College of Natural Sciences will host its annual Undergraduate Research Forum on Friday, April 8, 2011 in Welch Hall.
All CNS undergraduate students who have participated in research with CNS faculty or with faculty in other colleges or at other institutions (e.g. summer REU participants) are encouraged to present. The event will feature poster presentations by our top student researchers as well as cash awards for research excellence.
Interested students should first fill out an "Intent to Submit" form on the Student Participants page of the web site above. This connects them to resources and information, including the four workshops we sponsor to help students put together a poster and prepare to present.
Reminder: All undergraduates in the Ellington lab are expected to present their research. This doesn't apply to the freshman who have just entered the stream, but those who work over the summer will be expected to present next spring.
For full list of workshops and more information visit:
http://cns.utexas.edu/research/undergraduate-opportunities/undergraduate-research-forum/workshops
New Lab Hours
The lab is now open 9am-9pm Mon-Thurs, 9am-7pm Fri, & 11am-5pm on weekends.
Someone else MUST be in the lab with you, while you work. This person may be in the Aptamer Stream or in another stream.
Currently, the mentor schedule is the same, although we may switch that around,too. We'll let you know if changes are made.
Please do your "difficult" work when a mentor is present in lab, so he/she can help you.
Best wishes,
Gwen
Someone else MUST be in the lab with you, while you work. This person may be in the Aptamer Stream or in another stream.
Currently, the mentor schedule is the same, although we may switch that around,too. We'll let you know if changes are made.
Please do your "difficult" work when a mentor is present in lab, so he/she can help you.
Best wishes,
Gwen
RNA Bead Based Protocol Errors
Target Immobilization: Step 5. Denaturing should be @ 65C instead of 95C.
Eluting and Precipitating the RNA: Step 11. Resuspension should be in 10uL of diH2O not 20uL.
Reverse Transcription: Step 3. This step describes a component of the reaction mixture listed in step 4. I found this confusing when I was first perfroming selections. I think this step should be removed and the RNA template reactant should be added to the Rxn mixture list in step 4.
These are the errors that a few mentors and I knew of. If anyone else finds anything else please add a comment to this post.
(week 10 protocol)
Eluting and Precipitating the RNA: Step 11. Resuspension should be in 10uL of diH2O not 20uL.
Reverse Transcription: Step 3. This step describes a component of the reaction mixture listed in step 4. I found this confusing when I was first perfroming selections. I think this step should be removed and the RNA template reactant should be added to the Rxn mixture list in step 4.
These are the errors that a few mentors and I knew of. If anyone else finds anything else please add a comment to this post.
(week 10 protocol)
Announcements - 3/22/11
1. Selection targets have been assigned (check email). Please email me personally if you have a problem with the target you were assigned. And - MBP folks, continue with your practice selections & your target will be ready next week.
2. Due dates*:
a. Target discovery assignment + progress report (assignment posted) - April 3rd
b. Progress report - April 24th
c. Final report (assignment posted) - May 5th
* These dates differ from those on the syllabus. I wanted to give you a little bit more time to dig into the research before generating a progress report.
3. Protocols are posted on blackboard. Please post any protocol corrections here on the blog.
4. Assignments (i.e. Target discovery & progress reports) are posted on Blackboard.
Make sense? Post questions here.
Thanks,
Gwen
2. Due dates*:
a. Target discovery assignment + progress report (assignment posted) - April 3rd
b. Progress report - April 24th
c. Final report (assignment posted) - May 5th
* These dates differ from those on the syllabus. I wanted to give you a little bit more time to dig into the research before generating a progress report.
3. Protocols are posted on blackboard. Please post any protocol corrections here on the blog.
4. Assignments (i.e. Target discovery & progress reports) are posted on Blackboard.
Make sense? Post questions here.
Thanks,
Gwen
Reference Letter
Hello Gwen,
I was just checking on the status of my fellowship application, and I noticed that it indicated that they have not received your reference letter yet. I was wondering if you had to submit one since you'll be the one deciding?
Thanks!
Rose Wu
I was just checking on the status of my fellowship application, and I noticed that it indicated that they have not received your reference letter yet. I was wondering if you had to submit one since you'll be the one deciding?
Thanks!
Rose Wu
Plan - week of March 21 - 27, 2011
One more time ...
Please retake the quiz,
https://spreadsheets.google.com/viewform?formkey=dEctZ3lMOUdLSjNSd0FnNFRkdzUtZkE6MQ I'm sorry. The names didn't come across, so take it one more time, but enter in your name this time. I apologize for the inconvenience. Please take the quiz ASAP, so I can complete the target assignment.
Here's the plan for the week & how the targets will be assigned:
1. If you aren't enjoying the class and/or you want an "easy" target, then you'll stick with the lysozyme target. Lysozyme is a fun target! It'll make life a little bit easier for you & you get to see what it's all about to find an aptamer. Plus, you can hone your techniques and maybe feel a bit better about bench work in the long run.
Lysozyme is in the lab & you should be ready to run with these selections.
2. If you're unsure or somewhere in the middle, then you'll select against fibrinogen. This target has been selected against in the past, but an aptamer isn't necessarily almost guaranteed like with a lysozyme selection. Also - it's easier to understand the application of such an aptamer (i.e. easier than lysozyme aptamer application).
Fibrinogen in in the lab & you should be ready to run with these selections.
3. "Bring it!" If you're ready for a challenge, then you get maltose binding protein (MBP). Alex Miklos in the Ellington lab could use an aptamer to help him identify the "open" and "closed" forms of the protein. Here's what he said about it, "in pursuit of a surface-display system that I can use to evolve small-molecule binding proteins, an aptamer that would bind only to the closed form of a binding protein could prove to be useful (and probably publishable if nobody's done anything quite like this before)." Yes, the application of such an aptamer is a little bit more difficult to understand, so Alex will stop by & talk to us to get the point across.
The catch - MBP isn't ready yet. It won't be ready until the end of this week or early next week. So - if you get MBP, then you need to continue with your current selection until MBP is ready. Use the time to hone your techniques & learn some more about the protein.
Questions? Please post questions on the blog.
Thank you & see you this week!
Gwen
Please retake the quiz,
https://spreadsheets.google.com/viewform?formkey=dEctZ3lMOUdLSjNSd0FnNFRkdzUtZkE6MQ I'm sorry. The names didn't come across, so take it one more time, but enter in your name this time. I apologize for the inconvenience. Please take the quiz ASAP, so I can complete the target assignment.
Here's the plan for the week & how the targets will be assigned:
1. If you aren't enjoying the class and/or you want an "easy" target, then you'll stick with the lysozyme target. Lysozyme is a fun target! It'll make life a little bit easier for you & you get to see what it's all about to find an aptamer. Plus, you can hone your techniques and maybe feel a bit better about bench work in the long run.
Lysozyme is in the lab & you should be ready to run with these selections.
2. If you're unsure or somewhere in the middle, then you'll select against fibrinogen. This target has been selected against in the past, but an aptamer isn't necessarily almost guaranteed like with a lysozyme selection. Also - it's easier to understand the application of such an aptamer (i.e. easier than lysozyme aptamer application).
Fibrinogen in in the lab & you should be ready to run with these selections.
3. "Bring it!" If you're ready for a challenge, then you get maltose binding protein (MBP). Alex Miklos in the Ellington lab could use an aptamer to help him identify the "open" and "closed" forms of the protein. Here's what he said about it, "in pursuit of a surface-display system that I can use to evolve small-molecule binding proteins, an aptamer that would bind only to the closed form of a binding protein could prove to be useful (and probably publishable if nobody's done anything quite like this before)." Yes, the application of such an aptamer is a little bit more difficult to understand, so Alex will stop by & talk to us to get the point across.
The catch - MBP isn't ready yet. It won't be ready until the end of this week or early next week. So - if you get MBP, then you need to continue with your current selection until MBP is ready. Use the time to hone your techniques & learn some more about the protein.
Questions? Please post questions on the blog.
Thank you & see you this week!
Gwen
Selection Target Assignment
I'll assign aptamer selection targets this week. To help me best match the target with your skill set, preference, and to better enjoy this overall "selection" experience, please complete this simple 2 question quiz (15 seconds max). Please complete this quiz by Tuesday, March 22.
https://spreadsheets.google.com/viewform?formkey=dEctZ3lMOUdLSjNSd0FnNFRkdzUtZkE6MQ
Thank you,
Gwen Stovall
https://spreadsheets.google.com/viewform?formkey=dEctZ3lMOUdLSjNSd0FnNFRkdzUtZkE6MQ
Thank you,
Gwen Stovall
TriLink Modified Incorp
If you are considering DNA selections and think modified nucleotides may be of interest, I just got this e-mail from TriLink:
Q - Which of your modified nucleoside triphosphates can be incorporated into DNA molecules through PCR?
A – Aminoallyl-dNTPs, biotin-AA-dNTPs, 2-amino-dATP, 7-deaza-dGTP, and 7-deaza-dATP, 5-Methyl-dCTP, 5-Iodo-dUTP, 5-Bromo-dUTP, 5-Fluoro-dUTP, N4-methyl-dCTP, 5-propynyl-dUTP and 5-propynyl-dCTP, 2-thio-dTTP, 4-thio-dTTP and alpha-thio-dNTPs can be incorporated through PCR. As not all of these analogs will incorporate with similar efficiencies in PCR, some optimization may be needed. We recommend performing some initial PCR experiments using natural:modified dNTPs in ratios such as 1:0, 3:1, 1:1, 1:3 and 0:1 to identify the best conditions for modified nucleotide incorporation with robust amplicon yield.
In addition, 2'OMe-dATP and 2'Fluoro-dCTP can also be incorporated using a modified taq polymerase called Therminator III from NEB. Just ask Mimi. It needs to be optimized, but does work. 2'OMe-dA does not work as well though.
(hint, just click on the image above to see the whole thing)
Methods: A 10cm 10% Native Acrylamide self poured into ready gel housing with long wells was used. 10ul sample + 2ul 5X BioRad dye (Cat # 161-0767) was loaded onto gel. 25 cycles PCR with 45 second steps. 0.1 pmol target.
Observations: 10% Native Acrylamide Gel is easy to pour and takes only 25 minutes to run on these rigs. This gel ran for about 25 minutes at 250V and the darker bromophenyl blue was 1cm from the bottom. The 70bp amplified products midway through the gel with all polymerases tested.
Results: A 70bp product was detectable on the gel for all polymerases with normal nucleotides. However, there were also artifact bands detectable suggesting mispriming or inappropriate cycling conditions. Vent seemed to work the best with the cleanest darkest band for this template using 2’Fluoro dC. Therminator produced artifacts with 2’F dC also. Therminator also produced some smaller termination products with 2’Ome dA seen in lane 3 and 4. Taq polymerase could not incorporate any unnatural nucleotides and also did not show the characteristic banding pattern with natural nucleotides.
Conclusions: The thermo taqs (Therminator, Vent and Vent (-exo) produced banding artifacts with the normal set of nucleotideshese could largely be reduced with modified nucleotide 2’Fluoro-dC. . Therminator, Vent and Vent (-exo) were able to incorporate 2-F dC. None of the polymerases were able to adequately incorporate 2’Ome dA to a sufficient level detectable on this gel. 2’O-Me dA was not a good nucleotide for incorporation enzymatically. For future experiments, use 2’F dC with Vent for incorporation.
Q - Which of your modified nucleoside triphosphates can be incorporated into DNA molecules through PCR?
A – Aminoallyl-dNTPs, biotin-AA-dNTPs, 2-amino-dATP, 7-deaza-dGTP, and 7-deaza-dATP, 5-Methyl-dCTP, 5-Iodo-dUTP, 5-Bromo-dUTP, 5-Fluoro-dUTP, N4-methyl-dCTP, 5-propynyl-dUTP and 5-propynyl-dCTP, 2-thio-dTTP, 4-thio-dTTP and alpha-thio-dNTPs can be incorporated through PCR. As not all of these analogs will incorporate with similar efficiencies in PCR, some optimization may be needed. We recommend performing some initial PCR experiments using natural:modified dNTPs in ratios such as 1:0, 3:1, 1:1, 1:3 and 0:1 to identify the best conditions for modified nucleotide incorporation with robust amplicon yield.
In addition, 2'OMe-dATP and 2'Fluoro-dCTP can also be incorporated using a modified taq polymerase called Therminator III from NEB. Just ask Mimi. It needs to be optimized, but does work. 2'OMe-dA does not work as well though.
(hint, just click on the image above to see the whole thing)
Methods: A 10cm 10% Native Acrylamide self poured into ready gel housing with long wells was used. 10ul sample + 2ul 5X BioRad dye (Cat # 161-0767) was loaded onto gel. 25 cycles PCR with 45 second steps. 0.1 pmol target.
Observations: 10% Native Acrylamide Gel is easy to pour and takes only 25 minutes to run on these rigs. This gel ran for about 25 minutes at 250V and the darker bromophenyl blue was 1cm from the bottom. The 70bp amplified products midway through the gel with all polymerases tested.
Results: A 70bp product was detectable on the gel for all polymerases with normal nucleotides. However, there were also artifact bands detectable suggesting mispriming or inappropriate cycling conditions. Vent seemed to work the best with the cleanest darkest band for this template using 2’Fluoro dC. Therminator produced artifacts with 2’F dC also. Therminator also produced some smaller termination products with 2’Ome dA seen in lane 3 and 4. Taq polymerase could not incorporate any unnatural nucleotides and also did not show the characteristic banding pattern with natural nucleotides.
Conclusions: The thermo taqs (Therminator, Vent and Vent (-exo) produced banding artifacts with the normal set of nucleotideshese could largely be reduced with modified nucleotide 2’Fluoro-dC. . Therminator, Vent and Vent (-exo) were able to incorporate 2-F dC. None of the polymerases were able to adequately incorporate 2’Ome dA to a sufficient level detectable on this gel. 2’O-Me dA was not a good nucleotide for incorporation enzymatically. For future experiments, use 2’F dC with Vent for incorporation.
Beckman Scholarship Opportunity
Does research interest you ?
Do you think you would like to become a research scientist ?
Would you like a taste of what it's like in graduate school without the lectures or exams ?
Would you like to be in a program designed to maximize your research experience AND be paid to do it ?
Since 1999, the Dept of Chemistry & Biochemistry has run a Beckman Scholar program, one of a very small number of programs in the country that supports extended periods of undergraduate research. This program is now open to any UT undergraduate student, in good academic standing, who is a US citizen or permanent resident. As you will be expected to perform scientific research with one of our list of Beckman mentors (professors), applicants would be expected to have some scientific background, at a minimum, credit or registration in CH302. It is generally expected that students in their Junior or Sophomore year will be most suitable for this program; however students at any stage of their undergraduate career may apply provided they do not intend to graduate before Spring 2011.
We are funded to support two Beckman Scholars this year. Scholars will be paid to carry out full-time research in Summer 10 and 11 and part-time research in Fall 10 and Spring 11. The scholarship also include funds for research expenses and student travel to scientific meetings.
The time line for applications to this program is short. Our website contains all the information you need to know. Once you have read the website, complete the online application form, linked to our website, download the list of required supporting material and complete your application by noon on Monday March 28th, 2011.
http://research.cm.utexas.edu/beckman/
Do not think "Oh I'd never win a scholarship" or "These scholarships are already targeted for those students who win all the other scholarships", because you may be WRONG! While GPA is considered, it is by no means the main factor in our selection process. A strong enthusiastic interest in research and an aptitude for original thought are the most important things that you will need. In addition to choosing Beckman Scholars, we also attempt to place most finalists and many semi-finalists in research groups and nominate applicants for other scholarships.
Do you think you would like to become a research scientist ?
Would you like a taste of what it's like in graduate school without the lectures or exams ?
Would you like to be in a program designed to maximize your research experience AND be paid to do it ?
Since 1999, the Dept of Chemistry & Biochemistry has run a Beckman Scholar program, one of a very small number of programs in the country that supports extended periods of undergraduate research. This program is now open to any UT undergraduate student, in good academic standing, who is a US citizen or permanent resident. As you will be expected to perform scientific research with one of our list of Beckman mentors (professors), applicants would be expected to have some scientific background, at a minimum, credit or registration in CH302. It is generally expected that students in their Junior or Sophomore year will be most suitable for this program; however students at any stage of their undergraduate career may apply provided they do not intend to graduate before Spring 2011.
We are funded to support two Beckman Scholars this year. Scholars will be paid to carry out full-time research in Summer 10 and 11 and part-time research in Fall 10 and Spring 11. The scholarship also include funds for research expenses and student travel to scientific meetings.
The time line for applications to this program is short. Our website contains all the information you need to know. Once you have read the website, complete the online application form, linked to our website, download the list of required supporting material and complete your application by noon on Monday March 28th, 2011.
http://research.cm.utexas.edu/beckman/
Do not think "Oh I'd never win a scholarship" or "These scholarships are already targeted for those students who win all the other scholarships", because you may be WRONG! While GPA is considered, it is by no means the main factor in our selection process. A strong enthusiastic interest in research and an aptitude for original thought are the most important things that you will need. In addition to choosing Beckman Scholars, we also attempt to place most finalists and many semi-finalists in research groups and nominate applicants for other scholarships.
My Transcription Reaction Failed ... Now What?
Hi Gwen,
First, sorry to bother you on the weekend!
My group performed the PAGE protocol today and we did not get a band. I know that we're supposed to run the lsPCR products on an agarose gel to confirm that we actually had DNA to begin with, but this morning when I was adding the DNase to the transcription reaction, I accidentally added the DNase to the leftover lsPCR reaction instead. So now we have no PCR product.
What should we do now? If need be, we have no problem coming back next week and redoing the experiment.
Also, on the questions at the end of the protocol, number 4 asks "What was the concentration of RNA in uM at the end?" How would I do that? Should I just do a theoretical case and show how to do the calculations?
Again, sorry to bother you on the weekend.
Thank you,
xxxx
First, sorry to bother you on the weekend!
My group performed the PAGE protocol today and we did not get a band. I know that we're supposed to run the lsPCR products on an agarose gel to confirm that we actually had DNA to begin with, but this morning when I was adding the DNase to the transcription reaction, I accidentally added the DNase to the leftover lsPCR reaction instead. So now we have no PCR product.
What should we do now? If need be, we have no problem coming back next week and redoing the experiment.
Also, on the questions at the end of the protocol, number 4 asks "What was the concentration of RNA in uM at the end?" How would I do that? Should I just do a theoretical case and show how to do the calculations?
Again, sorry to bother you on the weekend.
Thank you,
xxxx
Summer Fellowship Application Deadline Approaching
Applications for CNS/FRI Summer 2011 Research Fellowships are now being accepted. This summer fellowship support allows current FRI undergraduates, FRI alumni, and undergraduates not affiliated with FRI to work full or part time in a research group for a minimum of 8 weeks in the summer. Current FRI students (in their first summer at UT) will be funded to work in their FRI Stream. FRI alumni and students not affiliated with FRI can be funded to work in another research group on the UT campus.
Find more information and a link to the application here:
http://cns.utexas.edu/honors-scholarships/scholarships-fellowships/research-fellowships
The deadline for applications is March 22, 2011.
Find more information and a link to the application here:
http://cns.utexas.edu/honors-scholarships/scholarships-fellowships/research-fellowships
The deadline for applications is March 22, 2011.
Looking for Ways to Pay for Study Abroad?
Phi Kappa Phi Study Abroad Grants Deadline 4/1
Phi Kappa Phi Study Abroad Grants are designed to help support undergraduates as they seek knowledge and experience in their academic fields by studying abroad. Forty-five $1,000 grants are awarded each year. These awards are available to all University of Texas at Austin students, not just Phi Kappa Phi members. Students at The University of Texas at Austin are also eligible for a $500 chapter Study Abroad Grant. For complete instructions, eligibility requirements and application forms, please visit http://www.utexas.edu/ugs/pkp/study-abroad-grants. All applications must be postmarked by April 1, 2011. If you have any questions, please contact the chapter at PKP@austin.utexas.edu.
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