Sequencing

For those sequencing pools, here are some guidelines and an FAQ from the Core Facility in MBB. After many discussions and failed reactions, here is what I typically do:

  1. First you must PCR amplify your pool to add new "A" at the end by Taq polymerase. Just run the pool with 10 cycles under normal conditions.
  2. Follow the general TA cloning kit instructions by Invitrogen to ligate your template into the pCR 2.1 vector with T4 ligase. Clone the ligated plasmid vector into Top10 cells and plate onto LB+Amp plates with X-gal.
  3. Clone check at least 11 white colonies/plate with M13F/R primers. I typically do multiples of 8 or 12 and run 7 or 11 down a gel with a ladder. Try to set up volume transfers to use multichannel pipettors where appropriate.
    1. Pick the colony with a pipette tip, shake in 1.5ml liquid LB + Amp then add to a 25ul PCR reaction - Grow overnight
    2. Run the PCR at 50C annealing for 25 cycles.
    3. Run down a gel and analize the correct size bands
    • (for our pools it will be ~200bp - incorrect, ~300bp - correct)
  4. Clean up the PCR reactions with a general silica filter column PCR cleanup kit (2-24 reactions fit easily into the centrifuges) or with a Millipore MultiScreen PCRµ96 plates (cat# LSKMPCR10) in the Ellington lab (>24 reactions).
    1. Add the PCR reactions to unused wells.
    2. Put the plate on a Qiagen QiaVac96 & use a water pull vacuum to remove salts & sol’n from reactions. Pull vacuum until wells are dry.
    3. Elute clean PCR reactions by adding 50 uL of water to the plate, shake plate for 5-10 minutes at room temp, pipet up & down to resuspend products, then holding the plate at an angle transfer the solution above the filter into a clean labeled tube.
    4. Make a 1:10 dilution of your reactions (brings most down to ~10ng/ul and spec if necessary).
    5. In a 1.5ml tube, add 1ul of diluted template (10ng total) + 1ul of 10uM primer (10pmol total) + 10ul diH2O
    6. Fill in coreweb.icmb.utexas.edu form and take over to the core.
      1. You only use one primer per sequencing reaction. Feel free to you use core primers but choose the one specific to the pCR 2.1 vector:
      • M13R (-24) 5'-GGAAACAGCTATGACCATG-3'
      • The T7 sequencing Primer (forward) 5'-AAATTAACCCTCACTAAAGG-3'
      • The M13 Universal Primer (-20) Forward 5'-GTAAAACGACGGCCAGT-3'
  5. Purify the plasmids from the liquid cultures that proved successful for long term storage using a mini plasmid prep kit. You can also spin the cells down, remove the supernatant and freeze the cells at -80C till the sequencing results return.
Notes:
  • I've found that sequencing cloned pool PCR products (~300bp) works very well when not too concentrated. I've actually had success from 1ng to 80ng template in the 12ul total reaction when the template is very small. Generally 1-10ng works great. Others have gone as low as 0.1ng target with 3pmol primer with success when higher concentrations failed. Above 50ng does not work well for PCR reactions for me. Cecil in the core recommends 5ng/100bp so 15ng/300bp fragments.
  • Primer quantities can be as low as 3pmol total.
  • The pCR 2.1 vector is 3.9kb and you must add your insert to the length when submitting plasmid for sequencing. A copy of the plasmid map can be found here.
  • Plasmid templates for sequencing work very well also, but extra time is involved waiting for the culture to grow overnight. When using plasmid template, 500ng total template is required per 12ul sequencing reaction. This is typically a 2-5 ul of the plasmid prep.
If the UT core facility does not work well for your targets, it could be their fault or it could be the cleanliness of your template. Here is an exhaustive link of many different sequencing houses: http://www.nucleics.com/DNA_sequencing_support/sequencing-service-reviews.html

In Houston, SeqWrite and Lone Star Labs are two notable labs.

Have I got a target for you ... Bisphenol A (BPA)

So - BPA is used to make plastics (i.e. it's found everywhere), but it causes negative health effects. Infants and fetuses seem especially vulnerable to BPA. This compound may be linked to obesity, thyroid problems, as well as a bunch of other stuff (suppresses DNA methylation, etc.). And - as a Mom with an 8 month old baby boy, I'm constantly trying to limit my son's BPA exposure by purchasing BPA-free items & minimizing his exposure to plastics (good luck). Anyhow - I think this might make a good aptamer target. An aptamer could be used to assay for BPA detection or possibly even used as an antidote to BPA exposure.

What do you think?

-Gwen

Should have 32 hours in lab

Many of you are falling far short of the required 10 hours/week. By now, you should've worked 32 hours. That's enough time to push through at least a couple of rounds of selection. At least 14 of you need more hours ... please don't wait until it's too late. Be nice to yourself & put in your hours at a steady pace, instead of trying to cram them in at the last minute. Cramming isn't good for the psyche.

Best wishes,
Gwen

Lab Notebooks

Hey guys,

We have only graded about half of the class' notebooks. If you have not had your notebook graded yet, please turn it in to Gwen or a mentor by the end of this week.

Thanks!

Be Safe

Follow UT guidelines today for classes. As it stands, all classes canceled, so NO LAB or LECTURE today. Will keep you posted when they open up things. Most of you are internet aware, so continue to follow information on Twitter, Fox News Stream and the news).

\UPDATE 12:20p: Campus is no longer under lock down, but all events canceled for the day. Good luck getting home/lunch. Again, no lecture or lab today...stay off campus and let the police do their work. Some interesting pictures coming out, if you have any send me the link. Here are some from Katherine. Former Aptamer stream mentor Arshia posted a video from Welch hall on CNN.

Stay safe and have a nice sunny day off school.

Questions about the database

What exactly am I supposed to be looking for when I'm searching for articles? I know on APS, you wrote we were only interested in the "original selection of aptamers," but does that mean the selections of specific aptamers or different selection techniques... or both? Are we primarily focusing on the process of selecting the aptamer or more so on both selecting the aptamer and the potential application for it, or even actual experiments where the aptamer has been used in a therapeutic/diagnostic capacity?

Aptamers Against the CD4 Protein Receptor for Inhibition of HIV Infection

The Human Immunodeficiency Virus, which is also known as HIV is a disease that compromises the immune system. Over time – in many cases, a long time – HIV slowly weakens the immune system until AIDS develops (1). AIDS stands for Acquired Immune Deficiency Syndrome. When a person has AIDS, his or her body has been weakened to the point where it is no longer able to effectively fight disease. As a result, many other health problems develop when a person has AIDS (1). HIV and AIDS has become an epidemic in many developing countries around the world and also claimed numerous victims in developed countries such as the United States.

HIV (types 1 and 2) enters susceptible cells either through binding of viral envelope glycoprotein (gp 120) to specific receptors on cell surface, mainly the CD4 molecule itself or through the beta-chemokine receptor-CCR5 (2). Other cells other than the helper T lymphocytes (CD4) such as some B cells, macrophages and glial cells of the central nervous system can also be infected by HIV so long as they bear the CD4 antigen. HIV belongs to a family of RNA viruses called retroviruses; so called because they possess a unique enzyme, reverse transcriptase, used to synthesize virus-specific double-stranded DNA from the viral RNA genome (3). The resultant DNA gets integrated into the genome of the CD4 where it may remain latent for a long time until activated. The DNA then is used as a template for RNA required for HIV production (2).


This immunopathogenic cycle can be prevented by production of a class of structural inhibitor molecules called 'aptamers'. Aptamers are short single‐stranded DNA or RNA sequences that are selected in vitro based on affinity for a target molecule (4). Aptamers also offer advantages over traditional antibody‐based affinity molecules in their ease of production, regeneration and stability; largely due to the chemical properties of nucleic acids versus amino acids. These oligonucleotide sequences have the capacity to recognize virtually any class of target molecules with affinity and specificity (4).With this capability, an attempt will therefore be made to develop aptamers specific to the target protein CD4 which are receptors for the HIV infection. Since HIV has inherently unstable epitopes (antigenic determinants) for antibody to bind to, it is believed that aptamers, known to be more superior inhibitors compared to antibodies could do that more effectively and thus prevent HIV infection.


References:

1. “About HIV and AIDS” AIDS Healthcare Foundation. http://www.aidshealth.org/?gclid=CJnN4orlo6QCFYlY2godJUhT6A. Updated: 2008

2. Chapel, H and Haeney M. Immunodeficiency-Immunopathogenesis of acquired immune deficiency syndrome, In: Essentials of Clinical immunology. London: Blackwell Scientific Publications. 1990:72

3. Okerengwo, A and Anyiwo C E. Immunopathology-The acquired immune deficiency syndrome, In: Essential Immunology. Port Harcourt: Pearl Publishers. 2006: 109-110

4. In Vitro Selection and Characterization of DNA Aptamers Specific for Phospholamban J. Pharmacol. Exp. Ther. (2009) 329(1): 57-63

Lab Notebooks

A new wave of panic and annoyance has permeated through the lab directed at the mentors about lab notebooks and grading. A few students were caught off guard when their lab notebooks were graded while in lab. In actuality, I said the first day (and it is in the syllabus) that lab notebooks would be graded more frequently this semester. It should be signed by a mentor weekly. It is 10% of your grade and your responsibility to keep updated (we will check 4 times during the semester). As a reminder (directly from the syllabus):

"A part of your grade will be based on what you put in your lab journal. A well-kept, organized, continually-updated lab book is what you are aiming for. You are required to write EVERYTING pertaining to experiments down in the lab notebook. This includes planning, protocols and procedures, results and interpretation, data, commentary and extra lab activities (racking tips, cleaning). Keep your notebooks in lab unless you take it home for updates or planning so that I can check it regularly. We do not require duplicate numbered pages, but understand that the notebooks are property of the lab and you must leave them with us after each semester. It is your responsibility to make duplicates of protocols and data you wish to keep. We recommend buying notebooks from the ACS student group (outside WEL 2.216), or the COOP (my preferred notebook is the Blue Line Composition, 192pg Cat# 69775-46003 $5.75 from the COOP). Make notebook entries often, discussing your plans, data and community involvement as a means to defend your contributions, attendance and ideas."

I also uploaded a rubric onto blackboard for you to see how we will be grading. But have no fear, this first go around is to show you what you are doing wrong. Don't freak out. I've instructed the mentors/TA to grade particularly hard this time so you know EXACTLY what to do. As Nia points out:

"I feel that at times [students] are perceiving that some things are okay in the FRI lab, [yet these same things] would not be okay in medical / graduate / schools and lab. We are trying to prepare [students] for their future by teaching them how to effectively keep a lab notebook [and document experiments]. I say it because having been taught and reinforced positive lab skills through FRI has helped me".

So, we are checking notebooks when you are around in lab. Keep them updated. Follow the rubric on blackboard.

Sign in/out

Just a reminder to students that when you sign in and out, don't fill in the boxes. Instead, just draw the box around the time you were in lab and in the middle write the total hours you were working. This will make it a lot easier to tally everyone's hours for the week.

Thanks!

Coats

We have noticed that there have been several instances where acrylamide or ethidium bromide drops have dropped on students and it seems to be that it is not understood that these chemicals are bad for you. Just because you can wipe them off does not mean that they stop being harmful.

We will begin reinforcing that you wear coats when working with these chemicals. This is not for our safety it is truly for your safety. You should become aware of the problems that can arise. If you do not trust us, look up the effects.

If someone asks you to put on a coat, please do so!

Thank you!

Where is the pipette gun??

The pipette gun seems to be missing, as of 9.22.2010. It, as well as it's charger, is MIA.

Posting data/results

Here's a short powerpoint presentation that might help when posting gels, pictures, charts etc.

Biotinylation of Targets

For the use of streptavidin coated microspheres, targets need to be biotinylated. Many targets we order are already functionalized with a binding moiety. If you have a smaller peptide or a protein that has not been functionalized and you wish to perform a bead based selection, you need to biotinylate your target now or in the future. Here is the protocol, and please speak with Brad about performing it the first time.


Broken Fish Tank!

On Saturday (or perhaps Friday night), someone left a message on the board informing us that the air pump had broken on the fish tank. I really do not want our fish to die, considering I almost already killed Jamal once (and again, I am extremely sorry). What should we do to remedy this problem?

Please Notify when we are low on primers!

Hi All,

If you are taking the last tube of Primers or notice that primers are going low, Let us mentors know so we can dilute more aliquots if you can not do it yourself!

Thanks!

Weekend of Sept. 18 and 19

Hello All! =D

I will be in lab tomorrow from 12-5 and Sunday 2-6. If you prefer me going in at a different time, let me know and I will try to make it =)

Come keep me company =)

The Aptamer Database

As a potential off bench project, I mentioned updating the aptamer database. The original publication should be read first to figure out what the database is all about. It hasn't been updated in nearly 4 years and is missing a wealth of current journals. A former member of the Aptamer Stream began updating it, but did not send me her final product and I've lost contact with her, so I guess we should start over again. Aptamer literature is fairly easy to search for on PubMed, but much more difficult to find exactly what you are looking for so having a database of all things aptamer is useful.

For an idea of what we can do with an up-to-date database, check out this article. Basically, we can mine the database for all sorts of information that isn't readily apparent by cursory glance. For instance, we could tell what the most common selection buffer is, or we could look for the most common 4 nucleotide sequence or the most common random region that produces aptamers. We could also find out if a skewed pool or random pool is more beneficial for screening aptamers.

Any of you can begin this project at any time. The first step is to get articles that aren't in the database. To begin finding primary articles, I would first suggest looking at a few of the reviews I've given you. In them are tables of selected aptamers with references. Look up those selections on pubmed and download the articles into Mendeley. Then you should find articles where people have used aptamers and find out the original aptamer publication for the particular aptamer. We are interested in the original selections of aptamers. The database does not include reviews, applications, patents etc. We may look to add these in the future. If you are off campus, you must append the following proxy address before the article: http://ezproxy.lib.utexas.edu/login?url= thus an article from NAR for instance would be: http://ezproxy.lib.utexas.edu/login?url=http://pubs.acs.org/doi/full/10.1021/bi1016233

I have created a Mendeley account for this purpose. Download Mendeley Desktop and use the account aptamer.streamut@gmail.com with the room number as the password. This way, we can keep the collection of pdf files synced up. It is easy to incorporate articles you find in the web using the Mendeley Bookmarklet. Just remember you have to be signed in and turn off your popup blocker for these sites. Then within Mendeley desktop if you double click the article it will download the PDF. I'm not a fan of Mendeley for PDF organization though after using Papers.app for Mac. In searching, a similar option is available for Windows called "I, Librarian". Haven't used or installed, but it seems cross platform and merges the best of Papers with the online collaboration of Mendeley. Sound off in the comments if you like I, Librarian.

Every time you go into Mendeley, sync with the server and then start searching for new stuffs. The sycn makes sure that anyone working on this project will have the most up to date links possible.

As for the database itself, you can and and modify citations from the website directly with the proper credentials. The database is set up like figure 1. Alternatively, we can grant you special access to the sandbox database through MySQL Admin, a graphical front end will be installed through phpMyAdmin, a graphical front end like MS Access that interfaces with the actual database and runs on your computer.
I have also created a webform in Google that allows you to add effectively add information for curation later.
Figure 1. Database table layout

If you find that the information should be displayed differently or that more information should be included, then feel free to tell me. For instance, there is another aptamer database called RiboaptDB and discussed in in this PDF. You may find they do things better or different than we do. Whereas they copied a lot from us, we may find they have implemented information searching or viewing slightly different that makes it easier to use. We can certainly re implement our site better or completely differently so please be aware.

You should also learn a bit about database setup. The aptamer database is hosted from MySQL on a linux virtual machine in the Ellington Lab. You should probably understand a bit about relational databased such as MySQL, so here is a nice tutorial from About.com. I have a complete copy of the database that can be imported into Microsoft Access if you want to play around with your own ways of adding information (i.e. making a new form or something).

Lastly, the project should include some type of website redesign and database input redesign. Using mendeley you should realize that Mendeley uses a database in the back end. If we can create some type of "referenced input" such as BibTeX or RIS format, we wouldn't have to input all the citation information manually. Additionally, if you know web programming (or know of someone who does) and want to try your hand at redesigning the site, please let me know. You could even interface it with some of the blogs or content management systems that are available if you know how to program PHP code. I can get you started on a scratch site to play around with before changes are moved over to the real site.

Tasks
  1. Find new literature - Articles detailing the original selection of aptamer binding species. You can also include different techniques, reviews or applications/uses of that aptamer in therapeutic/diagnostic capacity, but separate those into different folders in Medeley. We just want the aptamer sequence for now and the conditions it was selected under.
  2. Input all literature into Mendeley - You should label/organize into original selections, other catagories. We don't care much about the potential application for now. We also don't care much about
  3. Transfer literature from Mendeley to Database - probably an XML file of some sort.
  4. Add sequence information into database
  5. Redesign input method
  6. Redesign website
  7. Do cool things with the data

APP is in

APP came in today and is located in the FedEx box in my drawer. It needs to be resuspended, aliquoted and stored.

Proposal Information

Your target proposals are due today. Originally I had the date set as Tuesday, but I extended the due date.

You should have all the information you need to submit a winning proposal now. The original instructions for the proposal have been posted on Blackboard under Course Documents. When working on it, please take into account the comments and thoughts provided from members of the lab associated with your APS abstract.

For submission, I would like you to learn and use DropBox or WebSpace and provide a link from your APS abstract to the digital file of the proposal (in PDF or Word format). This will keep everything neat and prevent you from having to write something that long into the blog proper. DO NOT MAKE A NEW POST. Find your abstract, edit it with a line near the top that says something like, "The full proposal has been posted on DropBox" and link "posted on DropBox" with the link to your actual file. With the file in the cloud, any changes you make to it I will be able to see in real time. This also allows you to modify and edit the file without changing the link and I will always see the most up to date file. The same file can be used for your progress report and your final report with edits, additions and comments.

Instructions for using DropBox or WebSpace have been posted. As always, if you have problems or issues, please let me know.

-Brad

Aptamer Interpretive Dance

Well, this has seemingly gone viral through the interwebs today. It is an interpretive dance about aptamer selection. I laughed my butt off, but she has done a pretty good job. I would suggest more "interactions" of potential oligonucleotides and the "target" bullseye girl. But I like that the ones that interact all do the same dance and have the same color shirts. And check out the Thriller guy. Freaking awesome.

Selection of a DNA aptamer for homocysteine using SELEX from Maureen McKeague on Vimeo.

This PhD candidate has made it to the final 4 in the AAAS "Dance Your Ph.D." competition. To view the other finalists and see all of the 2010 entries, check out: http://gonzolabs.org/dance/.

There is even one from UT: http://vimeo.com/14503172. The longhorns look positively riveted! And the guy in the video you may recognise from Andy's lab as he is one of the administrative assistants.

New better magnets

I took some hard drives apart today and usurped the magnets inside. These magnets are very powerful and pull the iron beads to the side quite quickly (full removal within 2 seconds). You can even pull the beads completely out of the liquid (good for aspirating the entire wash volume and not disturbing beads!!!) Be careful with them though, they WILL pinch fingers and it DOES hurt. Eventually, I may affix them to an 80 well rack for easier use, or you are welcome to.

-Brad

Proper care and maintenance of targets

In general, proteins should not go through freeze thaw cycles. That means the -20 is not a good place to store them prior to aliquoting if they are already resuspended. If they come dry (desicated), then they can be stored in the -20. Otherwise, please place the targets that arrive in solution in the 4C fridge till we can aliquot them (that or the next day) and store them in the -80C. For the longer term storage, we need to aliquot between 50 and 400pmol/tube depending on the concentration and number of aliquots. These tubes will get a defined box within the -80C freezer with an informational note and also the same information should be posted on the APS.

As a rule of thumb, if the product comes on dry ice, place it in the -80. If it comes on freezer packs, then place it in the -20C and if it comes chilled but not frozen or without freezer packs then it is safe to place in the 4C fridge.

So, targets that we need to take care of currently are S100B for Emma and Kamaxi and MUC-1 for Sabeena...targets should start rolling in fast now.

S100B Is IN!!!!!!!!!

Hey guys, we just got S100B and I think it's Kamaxi's. It's stored in the -80 because the -20 goes through a ton of freeze/thaw cycles which isn't idea for the protein. The info for how to resuspend/use the protein is on the whiteboard under the Aptamer Stream section.

Sterile Technique

In the past couple of weeks I have noticed that many students (not only aptamer students) are not adherent to the previously established guidelines regarding sterile technique. Here are a few reminders:

1- Don't touch your "person" with your gloves on (this includes hair, clothes, skin)
2- Don't touch your "personal belongings" with your gloves (this includes phone, notebook)

This is important for your project because there are RNases everywhere -- if they interact with your "stuff" they will affect your results.

This is also important for yourself because there are toxins everywhere -- if you touch agarose (with ethidium bromide) or acrylamide with your gloves and then grab your phone you will take these toxins with you. Your hands might not currently be touching them but they will later on (imagine a little kid touching a toilet bowl and now washing his hands, he will carry his germs with him.)

Figures for Intro/Background

In the outline for the proposal, the intro/background section says there needs to be four figures in one place, and two figures in another place. I just asked Brad, and there needs to be four figures in the intro/background. Just thought I'd clarify for anyone else that had the same question.

Aptamer Research Meeting Schedule - Tuesdays @ 4:00

9/21/10 - Lindsay Becker presents
9/28/10 - Journal Club
10/5/10 - Michael Ledbetter & Sagar Kansara present
10/12/10 - Travis Hughes & Alec Rezigh & Mayank Aranke present
10/19/10 - Emma Weiss & Katherine Li & Amanda Romero present
10/26/10 - Journal Club
11/2/10 - Jialing Fang & John Ostrominski & Ashley Dawson present
11/9/10 - Kamaxi Patel & Damilola Olatayo & Sabeena Shaikh present
11/16/10 - Stephanie Philip & Austin Rezigh & Adaobi Anyiwo present
11/23/10 - Journal Club
11/30/10 - Lab Party

Aptamer Stream Flyer

I designed a stream flyer this year for the welcome picnic.  Feel free to print it in lab, e-mail it to friends, or I can give you a full color hard copy.  The purpose of this flyer is promotional, therefore if you have friends who are interested in the FRI or are in the process of stream sorting, be sure to pass this along.  Also, for those of you who are UGTAs for Research Methods, this can be of considerable interest.

Stream Flyer 2010

Wanna selection that works?

Previous FRI students selected against Fibrinogen and Hemoglobin S (sickle cell variant). These selections seemed to be working (i.e. 27% binding to Fib & maybe 4% binding to HbS).  The selections were originally using the N71 pool on filters with standard Hepes buffer.  If you're interested in picking up where previous student left off, then let Brad know. This might be a good project to do on the side or if you just need to feel good about your bench skills.

-Gwen

Here is some of the data from the former selections.  They were actually started quite a long time ago (during the pilot year - summer 2006) by two excellent students, Fan Fan Shen and Yuxuan Wang and are well labeled.  These two students began working on other projects during the fall of 2006 and did not follow up with these.  I just haven't had the format (like this blog) to formalize a continuation project out of it.  Figure 1 is the original binding assay showing good binding to both targets.  It is actually a good figure and I hope when you post binding assay data, you label it similar to these.

Figure 1.  Binding assay to Hemoglobin S and Fibrinogen performed late summer 2006.
Sequencing on these targets was performed for R6 (Fibrinogen - Figure 2) and R6/R10 for Hemoglobin S.

Figure 2. Sequencing data trimmed down to random region
Next steps include fully analyzing the sequences, performing individual binding assays on the clones, continuing the selection more rounds and analyzing further rounds for binding and individual sequences.

Attendance Logs - please write legibly

I'm recording your lab hours for the week. It isn't always easy to read the lab attendance sheet. Please write the total hours worked (not times worked) in the little boxes on the attendance sheet & refrain from writing me notes on the sheet ... although I do love to hear from you. If there's a major screw-up on the attendance sheet, then just draw a single line through the entry & send me an email (gwenstovall@mail.utexas.edu).

Also - please continue to ask Brad, Mimi, Holly, Xinh, Nia or me to sign-off on your hours. If a mentor isn't in the lab then please put "NM" below your time.

Questions? Let me know.

Thank you,
Gwen

Meeting Signup

Next week I will hold personal meetings with all of you. We will meet in lab and then either talk in lab our outside somewhere if it's nice. You should be getting an e-mail with the doodle poll link shortly.

Puddle

As you guys can tell there is a huge puddle outside of our freezer. Great news :: this can be avoided!

Don't keep the freezer door open for an extended period of time (i.e. when you're looking through a box, just remove the box and close the door.)

Don't continuously open the door (try to get everything you need in one turn instead of having to come back for more)!

Updates

Don't forget to keep updating the site on your progress/ problems/ difficulties/ successes or over all feel of getting back into lab! I remember when I came back after 1 month of being away and it was hard to get back in! Postponing will only make it harder to fit into your routine. Also, other people can offer insight into what is going wrong. It is not a great use of your time to stall the next step when you have plenty of people that have probably had the same problem!

Trust me that you will find more than lab help because you'll notice tons of people are in your classes so you'll find class help (which is how I got through physics and organic chemistry)!

Cloning and Sequencing of 1027 selection Part 1

I ligated and cloned the sequences (hopefully). Here's how the plates looked:


The later rounds have an alphabet after them (A/B) because during the summer, I split my total reaction into two selection rounds in the hopes of finding different aptamers. Both selections are done with the same parameters. The plates were ligated, cloned, and plated with the same parameters also. All four plates show large blue colonies surrounding by smaller white colonies with larger white colonies on the periphery of the satellite colonies.

The plates were treated with X-gal and Ampicillin, the former to track which cells had the Plasmid + insert and the latter to keep cells without the plasmid from growing. They are basically “weeding out” techniques that cut down sorting time. With the plasmid + insert, the colonies should grow white on an plate with X-gal and Ampicillin, but with only the plasmid, the colonies will grow a bright blue. The smaller white colonies could be Plasmid+ insert colonies, or they could be satellite colonies which are actually blue colonies in disguise around the larger blue colony which has absorbed all of the nutrients and materials (X-gal, ampicillin, ect) from the surrounding areas. The blue colonies are blue because they have taken up a plasmid without an insert and thus the lacZ gene is not disrupted so they are able to make B-Galactoside. Since the plates were made in July, it could affect how well the ampicillin works and since the X-gal was rolled on with colli rollers, the white splatters of colonies could be the effect of uneven spreading. The white colonies are smaller, perhaps, because with the insert, the genome is now larger and takes longer to copy.



This gel was run to test whether or not the white colonies had inserts in the plasmids since they were quite small and looked like they could be satellite colonies from the large, blue colonies. The results show that although there are multiple bands for some of the colonies, there are inserts at the 300BP mark, which is where the plasmid + insert should be. The colonies were randomly chosen and rubbed with a pipette tip to gather cells for the PCR reaction. Some of them show no insert at all which could be that they grew only after the large colony had eaten all of the ampicillin.

Aliquots and -20 Freezer Reorg

We have a few 6XO dye aliquots. Whichever mentors work today will make more.

I have reorganized the -20 freezer removing boxes from old lab members and moving around the aliquots. Everything now has it's own box instead of being left in open well racks for later knocking over. In addition, I put the general volumes/concentrations for aliquots. If we are running out, then please make at least 20 tubes more. Please don't let this happen to you.

There are a few tubes unaccounted for that will be thrown out at the end of the week. PLEASE check the second shelf to see if any of these are yours. Names on the 96 well PCR racks include: Adaobi, KL/VE and Alec, but I don't know who owns the actual tubes.

-Brad

6X EtBr Required!!

6X EtBr, we are in need of it! On a separate note, I am posting this information here with a certain degree of reservation. Are notifications such as this appropriate for the blog, or should they remain confined to the dry erase board at the front of the lab?

Lab Access Limited 9/14 in the evening

From 5p-7p 9/14, Emma McInturf, David Laude's RM TA, will need to have some portion of her class in PAI 214.  The lab will be open, but she get's first priority to the front two benches.  Hope this does not affect too many people.

Mentor Availability This Weekend Sept 11 and 12

Hi All =)

I will be in lab Saturday (Sept. 11) from 12-5 and Sunday (Sept. 12) 2-6.

Xinh

New 100bp Ladder Diluted and Functional

The new Invitrogen 100 base pair was tested during my R16 cycle course PCR.



The ladder can be seen here, along with third wash samples from six, nine, twelve, fifteen, and twenty cycles of PCR. As N58 RNA is slightly larger than 100 base pairs, its location just above the lowest ladder band is an indication of its effectiveness. Multiple 20 uL aliquots were created and placed in the -20 freezer. The stock ladder solution, previously diluted by Brad, was placed in the ladders and primers freezer box, distinguished by red tape.

Just getting Started

I was reading a pretty interesting post over at Lifehacker about getting started and how it is overrated.  I would have to agree/disagree.  I think for you all, just getting into lab and starting with your selections is the first step and should not be overlooked.  Just start doing it and you'll get back into it easy.  But, the point of the Lifehacker disagreement was more about long-term success.  These two points are suggested:
  1. Establish, over time, a deep emotional conviction that you want to follow the pursuit.
  2. Build an exhaustive understanding of the relevant world, know why some succeed and others don't, and know exactly what actions are required.
I would call #1 "Own and love your work" and #2 "Know your work".  Both are important to becoming successful.  I disagree with resisting the start because I think a lot of people, me included, need to get over that hump of starting, then fail a few times before we become truely proficient at a task.  So I have three suggestions for the Aptamer Stream and for college life in general.
  1. Just start on whatever target you want till you feel comfortable doing it.  Or start on the homework by reading the questions, then read the chapter.
  2. Be proud of your selection and target, own it.  Or be proud of the work you turn in for a grade.  If you have ownership, you will feel more responsible for the outcome and thus work diligently for the results.
  3. Know why other succeed at a topic, selection, etc and why some have failed.  Do the things that work for you.
  4. Understand the literature (goes beyond journal club).  Learn Learn Learn.  You will never stop learning, so use college to find out the best method that motivates your learning.
This has been a truth nugget from yours truly...

Acrylamide

There is new 8% acrylamide in the 4C fridge!

Nucleic Acid Aptamer Selection against TfR for the Therapeutic Treatment of Lysosomal Storage Diseases

Lysosomal Storage Diseases are inherited metabolic disorders that result from dysfunctional lysosomal enzymes. These enzymes facilitate the digestion of macromolecules within eukaryotic cells (Fong 2010). The absence of functional lysosomal enzymes results in the accumulation of macromolecules within the cell and can lead to many harmful effects on cells, tissues and organs (Futerman 2004). An efficient method of enzyme delivery could alleviate many of the problems associated with these diseases. The selection of an aptamer against a target membrane bound protein involved in endocytosis could provide efficient system of enzyme delivery to lysosomes. Post-selection modification of the aptamer would be necessary to link an enzyme to the aptamer. This method of aptamer-mediated endocytosis and post-selection modification could have a significant impact on the future of targeted enzyme delivery, as the design is very versatile.


Figure 1. Transferrin receptor-mediated endocytosis of Fe3+.

The transferrin receptor (TfR) is a membrane bound protein that facilitates the transport of iron into cells (Ray 2010). The binding of transferrin-Fe+3 to TfR signals for endocytosis of the entire molecule [1]. The molecule is transported to the endosomal compartment, iron is released and the rest of the molecule is incorporated back into the membrane. An aptamer will be selected against the transferrin receptor and modified post-selection with the addition of a functional lysosomal enzyme. This will enable the delivery of functional enzymes to lysosomes through aptamer-mediated endocytosis.

Though this form of enzyme replacement therapy is relatively new, there have been successful results, indicating that this therapeutic application of aptamers may be key to the future of lysosomal enzyme delivery. Researchers at UCLA have demonstrated the delivery of enzymes to cells deficient in lysosomal enzymes using the aptamer-mediated endocytosis method (Neufeld 2007). Chen et al. selected an aptamer against TfR and modified the aptamer with the attachment of α-l-iduronidase, a lysosomal enzyme. This aptamer complex was taken up by α-l-iduronidase-deficient mouse fibroblasts. The selection of an aptamer against TfR for aptamer-mediated endocytosis could have a revolutionary impact on the delivery of enzymes and treatment of various lysosomal storage disorders.

Human TfR from ARP- $259/ 50ug- http://www.biocompare.com/ProductDetails/1537096/Human-Transferrin-Receptor.html?

References

Chen, Chi-hong B. et. al. "Aptamer-based Endocytosis of a Lysosomal Enzyme." PNAS 105.41 (2008): 15908-5913.

Fong, Chin-To. "Lysosomal Storage Disorders." Merck & Co., Inc. Feb. 2010. Web. 04 Sept. 2010. .

Futerman, Anthony H., and Gerrit Van Meer. "The Cell Biology of Lysosomal Storage Disorders." Nature 5 (2004): 554-65.

Neufeld, Elizabeth F. "Aptamer Mediated Correction of Lysosomal Enzyme Deficiency." The Regents of the University of California. UCLA,

Ray, Partha, and Rebekah R. White. "Aptamers for Targeted Drug Delivery." Pharmaceuticals 3 (2010): 1761-778.

Image from: http://flipper.diff.org/app/pathways/info/1562


Files uploaded on blackboard

I have posted the first two lectures under "Course Documents" within blackboard. If you also didn't know I posted the Syllabus last week under "Syllabus".

Targets Ordered

The following targets have been ordered:
  • HIV-gag p24 (ordered last Monday) - Arrived 9-8-2010!!!
  • Dengue Virus Envelope (ordered last Monday)
  • S100B
  • INF-g
  • CXCL1
  • S100A4
  • APP - B-amyloid precursor protein
The following targets are already in lab
  • a-synuclein
  • EphA2
  • b-lactamase
  • transferrin
  • BPS 1027 and HCP1
  • Fluorescent proteins
I did not order MUC-1 or SATB1 because I have to talk with Sabeena and Lola (respectively) - or look at the comments on your abstracts and respond...

Potential Targets

Due to collaborations, I can't provide too much information here on the blog, but would be happy to discuss with anyone interested in the following targets to please see me.

FGF-8b
IL21R
Bcl-2
MMP-7

To functionalize a target

We typically use a biotinylated NHS ester that will bind to amines (R-N-H2 groups). Pierce makes the EZ-Link NHS-SS-Biotin. For this to work, you must have a target with an N-terminal amine (i.e every polypeptide) or a protein with an amino acid that contains amines (i.e. cysteine). The problem with functionalizing proteins this way is that you have to have the ratio of reagent to target just right to ensure you are attaching one or two biotins to the protein without wasting to much non-functionalized protein or biotinlyating the crap out of it. In addition, it can be difficult to assay the concentration properly.

Binding Assay 1 and 2

Binding Assay Data 1 Performed 6.21.2010
The first binding assay showed progressive binding through the rounds with a decrease at round 5 and an increase at round 7B. When restarting selection after a brief 2 week hiatus, it was discovered that the DNA in round 5 (1) had degraded into a short unusable strands and therefore, selection had to be restarted at round 3 and then progressed with parallel selections (A and B) per selection, per round. There is not a great amount of deviation from the average, which shows promise that the percentage binding is accurate. The green bar indicates the percentage of total binding species while the red bar indicates the total percentage of species that bind to things other than the protein.


Binding Assay 2 Performed 7.10.2010
This binding assay shows that with progressive rounds after round 7, there has been an increase in background binding, relative to the total binding. The assay for round 10 was not completed without problems due to an odd smear on the cartridge, despite multiple tries with different Storm Imagers and different cassettes. The cassettes and the screen were exposed for approximately 30 minutes each due to the high radioactivity of the P-32 used during the assay. Round 7 shows the most promise for the A round and will later be sequenced. When visualizing the cassette, there was such overexposure of the bottom where the nonbinders had washed out that it had essentially bleached the top part white. The pixel reader, however, can determine the differences of minute shades of pixels, so percentage binding can still be achieved. The original assay was supposed to be performed with rounds 7A, 8B, 8A, 10B, 10A, but due to the low yield after the etOH precipitation, it was impossible to continue. The top half of the cassette shows the binders for the assay with 1027 protein. The bottom half of the cassette shows that which was done without the 1027 protein. The blots in the top grid (1) cannot be seen, but based on the bottom grid (2). Grid 2 represents the washed away non binders. The darker blots on the bottom half of the grid show that more has washed out during the negative protein assay, which is promising. The green bars indicate percentage of total binding species and the red bars indicate percentage of total nonspecific binding species.


Lab this week?

Just wondering if we could work with target's, other than the one we had last semester, to get back in the swing of things. I was in lab today, but couldn't find any fibrinogen in the freezer (assuming we are out); can I practice on a different target (HBS and Angiotensin are still available) before I start work with my proposed one?

Figures in your proposals

Make sure to make a connection from the figures in your proposals to what you are saying. There should not be anything that "takes up space" it should have a purpose stated with your writing.

Ordering List

Running low on (edit as necessary):

SuperScript II Reverse Transcriptase

Therapeutic Development of an Aptamer against the Dangerous side of SATB1

Proposal Placed in Dropbox. The link is http://dl.dropbox.com/u/11745574/Proposal---Aptamer%20Stream%20%28September%202010%29%207.docx


DNA-Binding SATB1 (Special AT-rich sequence-binding protein-1) shown in Figure 1 (1) is a protein encoded in humans by the SATB1 gene. Its functions have been found to include double stranded DNA-binding, sequence-specific DNA binding, transcription factor activity and transcription repressor activity (1). The protein plays a role in the regulation of gene expression in the differentiation and activation of T cells. This makes it very important for an individual’s immune system (2). It has now been known to also play a major role in the development of aggressive breast cancer because of its ability to control the expression of thousands of genes, by reorganizing the chromatin organization and transcription profiles of breast tumors.
Figure 1:http://en.wikipedia.org/wiki/File:Protein_SATB1_PDB_1yse.png

In vitro studies that have been conducted showed a reduction in SATB1 expression in highly metastatic cell lines reduced the invasiveness of those cells as well as their capability for unattached growth. Aggressive cancer cells need unattached growth in order for them to move through the blood and lymph vessels. (2) This in fact proves that the inhabiting of SATB1 will hinder it from increasing the growth of aggressive cancer tumors. There are no known successful aptamers that have been developed as therapeutics against SATB1.

An aptamer if successful would be very useful as a therapeutic against the SATB1 protein. Inhibiting the functional pathway of SATB1 would prevent its reprogramming of the genome to change the expression of genes that encourage the metastasizing of breast cancer cells. With that said, the aim is to develop an aptamer with a high enough affinity of specificity to bind to SATB1 in order to prevent it from being a “master regulator”. Like most anti-cancer drugs any therapeutic development against SATB1 expressed by breast cancer cells will also have an adverse effect on normal cells. Researching what types of cells are likely to express SATB1 could be useful in terms of a preventative measure. In vitro selection would be the method used to select against SATB1. Figure 2 is an outline of what is discussed in this abstract.FIGURE 2) When Breast cancer cells express SATB1 protein, SATB1 reorganizes hundreds of genes thereby leading to the metastasizing of breast cancer cells. The inhibiting of SATB1 could prevent this from happening.

I came upon this on accident:

I was looking through CNN and this was in today's headlines, I though some of you might find it interesting. :)

Signing in/out

Do students need to be signed out by mentors?

Binding assay to determine binding affinity between N34 RNA and EphA2

Lack of binding between the ephrin A1 ligand and the EphA2 receptor, 130 kDa tyrosine kinase receptor found in adult human epithelial cells, causes unstoppable cell growth, and subsequently, development of tumors associated with epithelial cancers (Kinch, 2005; Ansuini et al, 2009). Inhibiting the kinase activity and phosphorylation of EphA2 protein receptor has been associated with a decrease in the growth of malignant cells (Ansuini et al, 2009). Ligand based approaches have been utilized by attaching a ligand to phosphorylating EphA2 protein which inhibits phosphorylation and subsequently decreases tumor growth (Walker-Daniels et al, 2002). A synthetic, highly modifiable ligand that can be used to inhibit EphA2 phosphorylation is an aptamer. Aptamers are nucleic acid fragments that have specific quaternary structures allowing them to bind with high specificity and affinity to certain protein targets, much like innate ligands, such as ephrin A1 (Phillips et al, 2008). Specific aptamers are filtered from a large nucleic acid pool through repeated, increasingly stringent tests, such as SELEX, which select for the highest affinity binders and amplification of those binders, and a binding assay can be performed to determine the progression of binding affinity through the rounds of selection.

A binding assay was performed to determine the affinity of filtered N34 RNA from R1, R3, and R5 to EphA2. 20 pmol of RNA, 40 pmol of EphA2 protein, and 200 pmol tRNA was used for the triplicate reactions which tested for both positive and negative binding. In order to detect the RNA, the double stranded DNA was labeled with radioactive beta 32P nucleotides during transcription. The radioactively labeled RNA was pipetted onto a nitrocellulose filter using the layout in figure 1. A nitrocellulose filter was used to filter the bound RNA and protein while a nylon filter collected the unbound RNA, as can be seen in figure 2.

Figure 1 Binding Assay Layout: Triplicate reactions were filtered using a nitrocellulose filter. To test for positive binding, R1, R3, and R5 protein and RNA was placed in three wells in the regions 1, 3, and 5 respectively. To determine how much of the binding was a result of RNA affinity for the nitrocellulose, R1, R3, and R5 RNA minus protein was placed in three wells in regions 2, 4, and 6 respectively.

Figure 2 Binding Assay: Nitrocellulose filter was used to separate the protein and bound RNA away from the unbound RNA which was collected on the nylon filter. The dots are a result from exposure of the 32P labeled RNA to phosphor plates.

Binding was minimal as shown by the faintness of the samples collected on the nitrocellulose filter in figure 2 and the comparison of the percent binding in figure 3. However, it must be noted that the results may be slightly skewed due to prolonged exposure between the 32P labeled RNA and phosphor plates which may account for the increased desensitization, and subsequently higher percent bound volumes, on the nitrocellulose filter. Nevertheless, the decreased average percentage of negatively binding RNA on the nitrocellulose filter (1.125% to 0.584%) does indicate the RNA with higher affinity for the protein, rather than the beads or filter, is gradually being selected. The average percent bound of high affinity RNA can be increased by progressively more stringent selection conditions to further reduce the amount of low affinity sequences and background binders.

Figure 3 Percent Binding: Percent binding is the amount of bound RNA and EphA2 on the nitrocellulose filter. It is calculated by dividing the total volume of protein and both bound and unbound RNA by the volume of bound RNA and EphA2.

Ansuini, H., et al (2009) “Anti-EphA2 Antibodies with Distinct In Vitro Properties Have Equal In Vivo
Efficacy in Pancreatic Cancer.” Journal of Oncology 2009: 1-10.

Kinch, M. S. (2005). Targeted drug delivery using EphA2 or EphA4 binding moieties. Patent No. 20050153923. Laytonsville, MD, US.

Phillips, J. A., et al (2008) “Applications of aptamers in cancer cell biology.” Analytica Chimica Acta 621 Review: 101-108.

Walker-Daniels, J., et al (2003) “Differential Regulation of EphA2 in Normal and Malignant Cells.” American Journal of Pathology 162: 1037-1042.

Don't cite Wikipedia

Please do not cite Wikipedia. Wikipedia is not a credible source and some of the information on the pages is wrong. You may use Wikipedia to find additional references. Read those references and then cite that, if appropriate.

List of questions:

How does one functionalize a protein?
How do you know what beads to use?
What is the significance to the different tags?
How do you know what beads to use? or columns? or anything else?
Does it matter what pool we use?
What is a good starting amount of pmol?
Do we need to start a new notebook, or can we use the old one?
Is the buffer they mention on the data sheet the one we should use?


*Reminder for Brad: Please send terrible binding assay results to Michael and I! :)

Volunteering for the Picnic

Please respond in the comments if you want to volunteer to work at the picnic. Information was posted here. So far I have Ashley and Katherine so far.