Bryan Hernandez/20.109/Lab notebook/Module 4/Day 2: Difference between revisions

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(New page: --~~~~ '''purpose:''' to try and optimize the protocol for finding gold binders from the first days experiment. also to prepare galactose culture tubes for tomorrow's lab. ==Protocol== ...)
 
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==Optimizing Gold Binding Yeast Screen Protocol==
--[[User:Bryanh|Bryanh]] 16:32, 2 May 2007 (EDT)
--[[User:Bryanh|Bryanh]] 16:32, 2 May 2007 (EDT)


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==Protocol==
==Protocol==
===Part 1: Optimizing panning conditions===
===Part 1: Optimizing panning conditions===
The only thing real ground rule here is that everyone will compare pCT-CON and pAu1. What experimental condition you vary in the panning protocol is up to you. The drawing below can be used to sketch your experimental set up. It may be possible for you to work on the other parts of today's experiments while the yeast are incubating with the gold.
'''expt1:''' This was identical to Day 1's procedure to screen for gold binders comparing PCT-CON to pAu1.  The only difference in this protocol was that instead of incubating the gold sheet in blocking buffer at room temperature and on a rocker that only moved in a circular motion we incubated it in a 30C incubator on a rocker that moved around in circles.  This change in protocol is aimed to reduce the number of bacteria that we see on out - control plate, that being PCT-CON by better blocking the Au surface.
[[Image:Macintosh HD-Users-nkuldell-Desktop-BE.109sixwelldish.png|thumb|left|375px| '''Six-well dish to help plan your experiment''']]
 
<br style="clear:both" />
'''Expt2:'''Also identical to the Day 1 protocol for screening yeast gold binders comparing PST-CON to pAu1. The only difference to this protocol was that we increased the time that we allowed the blocking buffer wash the surface of the gold before we added the yeast.  this would hopefully improve our negative control results as well by keeping non-specific binders from binding to the gold plate and resulting in high colony counts on our PCT-CON plate.


===Part 2: Library rescreen===
===Part 2: Library rescreen===
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===Part 3: Research proposal===
===Part 3: Research proposal===
Writing a research proposal requires that you identify an interesting topic, spend lots of time learning about it, and then design some clever experiments to advance the field. It also requires that you articulate your ideas so any reader is convinced of your expertise, your creativity and the significance of your findings, should you have the opportunity to carry out the experiments you’ve proposed. To begin you must identify your research question. This may be the hardest part and the most fun. Fortunately you started by finding a handful of topics to share with your lab partner. Today you should discuss and evaluate the topics you’ve gathered. Consider them based on:
discussed mad research ideas...
* your interest in the topic
* the availability of good background information
* your likelihood of successfully advancing current understanding
* the possibility of advancing foundational technologies or finding practical applications
* if your proposal could be carried out in a reasonable amount of time and with non-infinite resources
 
It might be that not one of the topics you’ve identified is really suitable, in which case you should find some new ideas. It’s also possible that through discussion with your lab partner, you’ve found something new to consider. Both of these outcomes are fine but by the end of today’s lab you should have settled on a general topic or two so you can begin the next step in your proposal writing, namely background reading and critical thinking about the topic. 
 
A few ground rules that are BE.109 specific:
*you should not propose any research question that has been the subject of your UROP or research experience outside of BE.109.  This proposal must be original. 
*you should keep in mind that this proposal will be presented to the class, so try to limit your scope to an idea that can be convincingly presented in a ten minute oral presentation. 
 
Once you and your partner have decided on a suitable research problem, it’s time to become an expert on the topic. This will mean searching the literature, talking with people, generating some ideas and critically evaluating them. To keep track of your efforts, you should start a wiki catalog on your OpenWetWare user page. How you format the page is up to you but check out the [[Yeast rebuild |“yeast rebuild”]] or the [[T7.2 | “T7.2”]]  wiki pages on OpenWetWare for examples of research ideas in process. As part of your “for next time assignment” you will have to print out your wiki page specifying your topic, your research goal and at least five helpful references that you’ve read and summarized.  




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[[Image: Exp2 pAu1.jpg|150px|Expt2 pAu1]]
[[Image: Exp2 pAu1.jpg|150px|Expt2 pAu1]]
results of both experiments are shown. in order the pictures are the pre-incubation results of: Expt1 PTC-CON, Expt1 pAu1, Expt2 PTC-CON, Expt2 pAu1.
It appears as though Expt2 was better at reducing non-specific gold binding.

Latest revision as of 13:58, 2 May 2007

Optimizing Gold Binding Yeast Screen Protocol

--Bryanh 16:32, 2 May 2007 (EDT)

purpose: to try and optimize the protocol for finding gold binders from the first days experiment. also to prepare galactose culture tubes for tomorrow's lab.

Protocol

Part 1: Optimizing panning conditions

expt1: This was identical to Day 1's procedure to screen for gold binders comparing PCT-CON to pAu1. The only difference in this protocol was that instead of incubating the gold sheet in blocking buffer at room temperature and on a rocker that only moved in a circular motion we incubated it in a 30C incubator on a rocker that moved around in circles. This change in protocol is aimed to reduce the number of bacteria that we see on out - control plate, that being PCT-CON by better blocking the Au surface.

Expt2:Also identical to the Day 1 protocol for screening yeast gold binders comparing PST-CON to pAu1. The only difference to this protocol was that we increased the time that we allowed the blocking buffer wash the surface of the gold before we added the yeast. this would hopefully improve our negative control results as well by keeping non-specific binders from binding to the gold plate and resulting in high colony counts on our PCT-CON plate.

Part 2: Library rescreen

Examine the Petri dishes that you spread last time. Count the number of colonies on each plate and enter your data in the table on the discussion page for this lab. It is OK to count only 1/2 or 1/4 or 1/8 of the colonies if they are densely growing. In these cases, try to find a representative sector to count and don’t forget to multiply for the total number of colonies on the plate. Circle four colonies that are nicely isolated from others on the "library" petri dish. Label those colonies "A" "B" "C" or "D".

Next you should set up four tubes with 2.5 ml of glucose-containing media and four tubes with 5 ml of galactose-containing media. Use colored dots to label the tops of each “A” “B” “C” or “D.” Use sterile technique since these will be used to grow any gold binding candidates for next time.

Finally use a sterile dowel to transfer some of the correct colony from your petri dish to the glucose-containing media, and balance the tubes on the roller wheel in the 30° incubator. Give the galactose-containing media to the teaching faculty who will innoculate them before next lab.You should also return to the teaching faculty the petri dish from the library screen so it can be stored until next time.

Part 3: Research proposal

discussed mad research ideas...


summary/interpretation

Expt1 PTC-CON

Expt1 pAu1

Expt2 PTC-CON

Expt2 pAu1


results of both experiments are shown. in order the pictures are the pre-incubation results of: Expt1 PTC-CON, Expt1 pAu1, Expt2 PTC-CON, Expt2 pAu1. It appears as though Expt2 was better at reducing non-specific gold binding.

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