Molecular Biology
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These are the protocols for the molecular stream.
Contents |
Spring 2007
Facebook Group: http://utexas.facebook.com/group.php?gid=2244160718
Where you can find all the Aptamer stream specific things for Spring 2007.
We strongly encourage you to use, and alter, the Wiki. It's fun and useful, a Wiki is pretty boring without your input, and you just might gain the confidence you need to inform Wikipedia in no uncertain terms that you favorite underground band released their third album in December, not November.
If you want to talk about the Wiki, or give feedback or ideas on the class in general, put it here! Feedback
If you want to post a notice to/chatter with your classmates (like "Stop making such a mess," or "I, like, totally have a crush on _____,") put it here! Stay civil, of course. Message Board
If you want to have a place to freely mess with the Wiki to learn how to do it, go here! Sandbox
Fall 2007
If you are registering for Ch 369K, you have to fill out a green "Intent to Register for CH 369K form. They can be found in the Chemistry Undergrad Office (Wel 2.212). If you are registering for BIO 377, you don't need to turn in a form before registration, but I would like you to fill out out before the start of classes in August. They are in ESB 1.
Turn the forms in to Brad Hall (leave near the sign-in binder in ESB 532) and they will be signed by Dr. Ellington.
From The Chemistry Undergraduate Advising Center:
"Due in part to the requirements and recomendations put forth by ACS for degree accreditation, students taking CH369K are required to turn in a report on or before the last class day of the semester for which they are registered in the course. A copy of the report with your faculty supervisor's approval must be received in the undergraduate course office WEL 2.212 on or before the last class day.
"Minimum requirements for report: Cover page + 6 pages of text (excluding tables and figures) + bibliography (or foot note page). The cover page should include the title, your name, your faculty supervisor's name and signature, the course number (CH369K) and the semester. The text length should be a minimum of 6 pages, typed and double spaced (12 point font and 1" margins). The recommended report style should be that of a research manuscript, but this is ultimately left to your research professor's preference."
Presentation Schedule
| Week 1 | Date | Schedule | |||
|---|---|---|---|---|---|
| 1 | Aug 31 | Lab introductions, Andy's projects | |||
| 2 | Sept 7 | Proposal requirements, other projects | |||
| 3 | Sept 14 | Brad in Indy, Andy conducts meeting, Yuxuan presentation | |||
| 4 | Sept 21 | Training History Due | Lisha S | OPEN | OPEN |
| 5 | Sept 28 | Target Assignment Due | Min-Jeong C | OPEN | OPEN |
| 6 | Oct 5 | Katelyn D | Ryan M | OPEN | OPEN |
| 7 | Oct 12 | Liz P | Nicole V | Justin L | Christina C |
| 8 | Oct 19 | Charles Z | Erin K | Akash P | David K |
| 9 | Oct 26 | Progress Report Due | Jeremy F | Lucy C | Mir A |
| 10 | Nov 2 | Caroline S | Angelica V | Wes B | Brian V |
| 11 | Nov 9 | Alex T | Habeeba N | Ben M | Yuxuan L |
| 12 | Nov 16 | Grace E | Sara Z | Jerrad S | Morgan M |
| 13 | Nov 23 | No Meeting: Thanksgiving Holiday | |||
| 14 | Nov 30 | Final Paper Due (Vishu A) | Travis S | Kathryn R | Anju G |
| 15 | Dec 7 | Archival, Finish up and Lab party | |||
Andy's Teams
Team 1. Select aptamers against nucleic acid-like targets.
- Design oligonucleotide sequences (pick length, composition, sequence).
- How do these variables affect the answers you're likely to get?
- Order the same oligonucleotide as both DNA and PNA (peptide nucleic acid), with a biotin moiety.
- What are PNAs, what do they look like, where would you get them from?
- Immobilize on streptavidin-coated beads.
- What is the basis for streptavidin-biotin interactions? Where would you get these beads from?
- Select RNA binding sequences based on a centrifugation partition.
- What are possible artefacts? What controls or simultaneous experiments could you run to prevent these?
- After multiple rounds, clone and sequence binding species.
- Compare binding motifs for DNA versus PNA.
- How will you identify binding motifs?
- Determine how many binding motifs you obtained; compare with how many binding motifs you expected.
- How will you calculate how many binding motifs were expected?
Further questions and issues:
- Has anyone done this before? What does the primary literature look like?
- PNAs can potentially be used as antisense drugs. How might your findings influence the design of these drugs?
- If you used a library that could be introduced into real-time PCR reactions, you could actually quantify the progress of the selection. What is real-time PCR? How does it work? How can it be used to quantitate amplicons?
- How could your data be used for calculating the delta G of binding of a given PNA base-pair?
Team 2. Select double-stranded DNA binding sites for transcription factors.
- Identify and order commercially available transcription factors.
- Where and how will you look for these (hint: you have to buy them, so why not Google 'transcription factor' and 'price' or 'catalog')? How will you figure out which transcription factors are interesting?
- Carry out filter-based selections using a double-stranded DNA library.
- After multiple rounds clone and sequence binding species.
- Identify binding motifs.
- How will you identify binding motifs?
- Compare binding motifs with those in the human genome.
- How will you do these comparisons?
Further questions and issues:
- Has anyone done this before? What does the primary literature look like?
- Can double-stranded DNA 'decoys' of transcription factors be used as drugs? How might this work? Has anyone done this?
- How could your data be used to find otherwise unknown transcription factor binding sites in a genome?
- How could your data be used to map transcriptional networks in a newly sequenced genome?
- How could you get data on multiple transcription fators simultaneously or in parallel?
Team 3. Select and compare aptamers under different conditions.
- Identify different buffer conditions you might use for selection.
- What are your variables? Ionic strength, pH, metal ions, temperature, what else? Which variables are most interesting or important to a successful selection?
- Select anti-lysozyme aptamers.
- Clone and sequence aptamers.
- Compare binding across different sets of conditions.
- Do aptamers that bind at one ionic strength or pH still bind at another? How specific is evolution for a given condition? Are some variables more important than others?
Further questions and issues:
- Has anyone done this before? What does the primary literature look like?
- Do the aptamers from a given pool or different pools bind at the same or different sites on lysozyme? How would you determine this? How might you do an ELISA? What is an ELISA?
- Do your experiments have any bearing on the origin of life? It is thought that modern life was preceded by a 'RNA world.' What is the RNA world hypothesis? Is a robust RNA world more likely under some environmental conditions than others? Did your experiments delimit the conditions under which RNA was more or less functional?
Protocols
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Sequencing Guidelines
[ICMB Core Sequencing] The core provides primers for free, but I've found them suspect. Therefore, I suggest providing your own primers.
Samples should be in 1.5 ml tubes with 500ng target and 10 pmol primer in 12ul total ul. This means if you run a PCR and see a nice band, it is probably around 50ng/ul and you are fine on the target. For the primer, add 0.5 ul of 20uM primer. For sequencing, these are typically M13F and M13R.
Individual samples are $4 and plates are $300.
Get the samples in by 10am to be processed that day.
Fill out the online request form (linked above). Label the tubes with your initials and the row number from the form (BH 1, BH2, BH 3, etc).
Take the samples to MBB 1.426 and put them in the green rack in the beer cooler (glass front fridge).
Before YOU Leave Lab Each Day
- CLEAN UP !!!!
- Parafilm - Throw away
- Goggles - Replace
- Trash - Throw away
- Glassware - Clean and dry
- Gel Rigs - Put all rigs, combs, spacers, places, etc...back where you found them
- Empty Tip Boxes - go on shelf under white board for refill
- Ice Buckets - dump out ice, place on center shelf
- Coliroller tubes - go on shelf for refill
- Lids - to prevent contamination, always put the lids back on tip and tube boxes.
Recipes
* Solutions
Notes and General Info
* Notebooks vs. Reports - How to present data
Literature Reading
Due to copyright laws, these documents are on blackboard...go there instead.
