yeast transformation failed after doing it many times - Molecular Biology
Transforming yeast with DNA is a very similar process to transforming E. coli, but with just enough differences to trip you up if you let your attention slip. Whether. Bacterial transformation is a common technique in molecular biology that every student of the life sciences should be familiar with. Transformation is the. Hi, I'm trying to transform S. cerevisiae with a 11Kb plasmid. I use the LiAc method, and this has always worked for me in the past. Recently however, I have .
Although it's more of a "my shittiest experiment" than "my shittiest method", as the method was technically fine. But you can join the party without invitation as well!
20.109(S07): Yeast transformation
So, here we go. Some Background At the time, I had a 7 weeks internship in a microbiology lab about which I wrote one of my first poststhe only post of mine ever to be curied! The ER or Endoplasmic Reticulum is a large part of eukaryotic cells - cells that have a nucleus so no bacteria!
It has several functions, making sure proteins are folded correctly is one of them because proteins usually need a 3-dimensional structure to function as intended. On the membrane of the ER, there are several channels that are responsible for transporting proteins into and out of the ER, because simple diffusion would be way too slow. In yeast, one of these channels is called SEC But why do we care? In humans, a similar channel in the ER-membrane exists. Proteins that are wrongly folded in the ER it's a biological system, there are always mistakes need to be transported to the outside and taken apart again, or the cell might die.
There are a lot of diseases, especially neurodegenerative diseases like Alzheimer's disease are linked to the aggregation of misfolded proteins. Knowing how the cell transports them and how to support or fix this system is thus something very interesting. And it's easier to test the basics in yeast, before doing weird stuff to humans, you know? The Experiment The insertion of the genes to overexpress the proteins we wanted to check into the "damaged" yeast strains was a piece of cake and worked without any further issues.
As such, it's perfect to monitor how a cell handles misfolded proteins. It can be almost anything, bacteria are usually transformed with a marker gene for antibiotic resistance.
Rough model of how the plasmid should look like My first step was to take some E. Then, the purified plasmids are transformed into the yeast 1.
As you can see, not every yeast cell absorbed a plasmid during this process. That's why we need the marker gene and the dropout medium: We only want yeast cells that have been successfully transformed.
In theory, my plate with the yeast was supposed to look like this after 3 days: The sad truth was But I was a 4th-semester student with not a lot of lab experience, I had probably just done something wrong!
Maybe the yeast hadn't properly absorbed the plasmid, after all, it was a foreign gene and yeast isn't so super happy about introducing foreign DNA into its cell. Close the cap and invert to mix, flicking in the last drops when the eppendorf is upside-down. Today you will use a kit sold by Q-biogene to prepare competent FY The contents of the kit are proprietary but the protocol seems most like ones for chemically competent cells this link leads to just one of many similar protocols.
Begin by harvesting 10 ml of log phase FY in a 15 ml conical tube. Spin these cells with a balance at rpm for 5 minutes in the clinical centrifuge. Remove the supernatant by aspirating. You do not have to remove every drop. Wash the cells with "wash solution" most likely just sterile water! You can resuspend the cells in the 15 ml conical in 3 ml of wash solution and then split this volume between three eppendorf tubes.
Harvest the cells in a microfuge be sure to balance your tubesspinning 1 minute at full speed. Resuspend each pellet in 50 ul of "competent solution" most likely lithium acetate and DTT which permeabilizes the yeast through an unknown mechanism. Unlike chemically competent bacteria, competent yeast are not "fragile" in this state and can remain at room temperature.
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Transformation Add 5 ul of your "no template" PCR reaction to one eppendorf and label the top appropriately. This should serve as your "no DNA," negative control.
Flick the tube to mix the contents. This plasmid bears a yeast origin of replication and a URA3 gene and will serve as a positive control for transformation. Add 5 ul of your "plus template" PCR product from last time.
This is your experimental sample. You can give the remainder of your PCR products to the teaching faculty who may run them on an agarose gel, depending on the outcome of these transformations. To each tube add ul "transformation solution" to your cells. This material, most likely polyethylene glycol "PEG" aka antifreeze is thick and goopy and is included in transformation protocols to help deliver the DNA into the yeast. Use your P to pipet the yeast and the "transformation solution" and vortex the tube to make an even suspension.
During this hour we will work on the Materials and Methods section of your upcoming lab report. If you can periodically "flick" your tubes to mix the contents, this will help keep the cells from settling to the bottom. After at least an hour longer is OK tooflick the tubes to mix the contents and then spread ul of each mixture on your SC-ura dishes, plating the experimental transformation twice.
For next time Write the Materials and Methods section for your lab report based on the material we've done so far, namely Primer design, PCR and yeast transformation. Again consult the writing instructions we've provided. Again, you and your lab partner can and should help eachother. When it comes time to write, you must do so on your own. You and your lab partner will hand in individual assignments. And again, please submit this part of the assignement electronically to both nkuldell and breindel AT mit DOT edu.
Read the relevant article by Wu et al, published in Mol Cell in There is also an associated review article that was written to celebrate and highlight this important work on the structure of the SAGA complex. You might find this review helpful but you are not required to read it. Below you will find some questions to guide your reading of the Molecular Cell article. You do not have to turn in the answers to these question.
You don't even have to answer them all! They are provided to help you decode and understand the most important and aspects of this paper that are relevant to your investigation and your upcoming writing assignment. Begin by skimming the whole article! This means read the summary once.
(S07): Yeast transformation - OpenWetWare
Read the first sentence of each paragraph of the introduction. Read the subdivision headings of the Results section. Look at all the figures and their legends. Read the first and last paragraph of the Discussion. Follow with a more detailed reading of the Introduction: Note in two or three words the topic of each paragraph of the introduction.
What do you think of the first sentence? Do you agree with what it says? Is it sufficiently broad? What about the second paragraph? Is it more detailed and relevant to the investigation than the first?
Is the of polypeptides what you expected? In the second paragraph the authors start to break the function of SAGA into functional modules. List them and the subunits involved.
What is a histone fold? If the subject of the second paragraph was functional composition, what is the subject of the third paragraph?
The fourth paragraph begins to address the genetic and biochemical evidence for how the modules of SAGA fit together. Next work on the Results: Next time we meet, you and your partner will be randomly assigned a portion of the text to describe to the class but try to understand it all, at least superficially.
A 3d model… a. Read this abstract and a little of the paper itself from the Nature Biotechnology link to get some idea of what TAP-tag purification is. How many domains were identified in SAGA? Mapping Taf subcomplex a. What does this suggest about their function? How were antibodies used in this section of the paper? Localization of Ada1 and Spt7 a. What was the goal of this series of experiments? What is the significance of this?