Difference between revisions of "20.109(F08): Mod 2 Day 2 Yeast transformation"
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In the laboratory, "controls" help answer the "what if" questions. They are equally or perhaps more important than your experimental samples and often more numerous. If the controls in an experiment haven't worked there is very little point in considering the data you have collected. Even experienced researchers often wish they had included a better or a different control for an experiment since data often leads to more questions, some of which might have been anticipated. | In the laboratory, "controls" help answer the "what if" questions. They are equally or perhaps more important than your experimental samples and often more numerous. If the controls in an experiment haven't worked there is very little point in considering the data you have collected. Even experienced researchers often wish they had included a better or a different control for an experiment since data often leads to more questions, some of which might have been anticipated. | ||
− | Your experiment today is straightforward: transform your yeast cells with your PCR product and look for cells that can grow on media lacking | + | Your experiment today is straightforward: transform your yeast cells with your PCR product and look for cells that can grow on media lacking tryptophan. What if you return to lab next time and saw no yeast colonies growing on any of your petri dishes? What if you return to find yeast covering the plates completely? How can you be sure these are even yeast and not bacterial colonies? You've transformed and transfected cells before...what controls were used for these experiments and can they be applied here? |
The <b>negative control</b> helps confirm that any positive experimental data is arising from the experimental sample and not some random event. For example, leaving the DNA out of a transformation reaction is a negative control. No colonies should grow from that sample. If colonies do grow with the negative control then perhaps the petri plates are contaminated, or perhaps the plates are the wrong kind, or perhaps the yeast are already able to grow on those plates without your experimental DNA. The negative control can't distinguish these possible explanations but it can tell you that something needs to be re-worked. | The <b>negative control</b> helps confirm that any positive experimental data is arising from the experimental sample and not some random event. For example, leaving the DNA out of a transformation reaction is a negative control. No colonies should grow from that sample. If colonies do grow with the negative control then perhaps the petri plates are contaminated, or perhaps the plates are the wrong kind, or perhaps the yeast are already able to grow on those plates without your experimental DNA. The negative control can't distinguish these possible explanations but it can tell you that something needs to be re-worked. | ||
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</center> | </center> | ||
− | It seems like this experiment "worked," really really well, but in fact the number of colonies on the experimental sample is too "good." The PCR product isn't expected to give rise to trp+ cells as often as plasmid DNA can. After all we're relying on recombination of the | + | It seems like this experiment "worked," really really well, but in fact the number of colonies on the experimental sample is too "good." The PCR product isn't expected to give rise to trp+ cells as often as plasmid DNA can. After all we're relying on recombination of the 39 bases flanking the product to integrate the <i> TRP1</i> gene. And if the SAGA-subunit deletion makes the cells even a little bit sicker than wild type, the correct product will be even harder to get. So how can this "too perfect" data be explained? |
Remember what is in that PCR sample: | Remember what is in that PCR sample: | ||
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*leftover Taq...not a problem | *leftover Taq...not a problem | ||
*template DNA...hmmm... | *template DNA...hmmm... | ||
− | What is to stop the <i>TRP1</i> template DNA, just another plasmid after all, from giving rise to cells that grow on SC-trp? To solve this problem the PCR product (expected to be around | + | What is to stop the <i>TRP1</i> template DNA, just another plasmid after all, from giving rise to cells that grow on SC-trp? To solve this problem the PCR product (expected to be around 1500 base pairs) could be purified from the template (around 4 or 5 Kb) using an agarose gel and a Qiagen kit as you did before. A more elegant solution though, is what you will use since the template you were given last time has no yeast origin of replication. Thus the yeast will not be able to copy the plasmid and it will be diluted then lost from your transformed samples. This "trick" has saved some time in the lab but did require some anticipation. Hopefully you can see the value. |
==Protocols== | ==Protocols== | ||
===Part 1: Concentrate your PCR product=== | ===Part 1: Concentrate your PCR product=== | ||
+ | Your PCR products are in a volume of 100 ul and we'd like to pool the two reactions that were done in the presence of template and concentrate the material in order to transform all of the product. We'll use a Qiagen column for concentrating the PCR'd materials. | ||
+ | #Move the contents of your PCR tubes into full-sized eppendorf tubes and add 5 volumes of '''PB''' (e.g. if you moved 100 ul then 5 volumes would be 500 ul). Recall '''PB''' is a high salt, low pH buffer that is added so the DNA in your reactions will bind the silica membrane. The salt in '''PB''' is guanidine hydrochloride, a chaotropic salt, meaning it will exclude water from the DNA, effectively precipitating it so it will bind the membrane in step 2. | ||
+ | #Get a QIAquick column and a collection tube from the teaching faculty then pipet the PCR/PB sample into the top. Microfuge the column in the collection tube for 60 seconds. Remember you must balance your tube in the microfuge. '''Note:''' The maximum volume for the columns is ~750 ul so you may have to pass the volume through the column in two spins. | ||
+ | #Discard the flow-through in the sink and replace the spin-column in the collection tube. Add 750 ul of '''PE''' to the top of the column and spin as before. Qiagen sells '''PE''' and does not reveal all its contents, but the solution is at least 80% ethanol which keeps the DNA precipitated and on the membrane but washes the salt away. | ||
+ | #Discard the flow-through in the sink and replace the spin-column in the collection tube. Spin for one more minute. Strange as it seems this is a very important step since it removes any residual ethanol from the membrane in the spin-column. If you forget this step the ethanol will elute with your DNA and will inhibit the upcoming transformation. | ||
+ | #Trim the cap off two new eppendorf tubes and label the side with the name of sample, your team color and the date. Place the spin-column in the trimmed eppendorf and add 20 ul of '''EB''' to the center of the membrane. '''EB''' is 10 mM Tris pH 8.5. Since the DNA will solubilize at low salt and high pH, it elutes from the column when '''EB''' is added. Lab water can also be used to elute the DNA but it s worth remembering that the pH is likely to be more acidic than 8.5 and so the company recommends eluting with EB when possible. Allow the column to sit at room temperature for one minute and then spin as before. The material that collects in the bottom of the eppendorf tube is ready to be checked on a gel and also transformed. | ||
===Part 2: Competent cells=== | ===Part 2: Competent cells=== | ||
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#Add 10 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. | #Add 10 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 500 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 P1000 to pipet the yeast and the "transformation solution" and vortex the tube to make an even suspension. | #To each tube add 500 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 P1000 to pipet the yeast and the "transformation solution" and vortex the tube to make an even suspension. | ||
− | #Incubate the tubes at | + | #Incubate the tubes at 30 for approximately one hour, along with 4 SC-trp petri dishes, with their lids ajar if there is moisture on their surface. During this hour you can: |
+ | #*add 2 ul of loading dye to the remaining volume of PCR products and load them on a 1% agarose gel to verify recovery. | ||
+ | #*work on the Materials and Methods section of your upcoming lab report. | ||
+ | #*periodically "flick" your tubes that are incubating at 30 to mix the contents. This will help keep the cells from settling to the bottom of the tube. | ||
#After at least an hour (longer is OK too), flick the tubes to mix the contents and then spread 250 ul of each mixture on your SC-trp dishes, plating the experimental transformation twice. | #After at least an hour (longer is OK too), flick the tubes to mix the contents and then spread 250 ul of each mixture on your SC-trp dishes, plating the experimental transformation twice. | ||
− | #Wrap your plates with your colored labeling tape and incubate them, media-side up in the | + | #Wrap your plates with your colored labeling tape and incubate them, media-side up in the 30 incubator until next time. |
DONE! | DONE! | ||
==For next time== | ==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 [[20.109(F08):Guidelines for writing up your research| 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 assignment electronically to both nkuldell and astachow AT mit DOT edu. | #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 [[20.109(F08):Guidelines for writing up your research| 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 assignment electronically to both nkuldell and astachow AT mit DOT edu. | ||
− | #Read the relevant article by [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15260971&query_hl=3&itool=pubmed_docsum Wu et al, published in Mol Cell in 2004]. There is also an associated review article [ | + | #Read the relevant article by [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=15260971&query_hl=3&itool=pubmed_docsum Wu et al, published in Mol Cell in 2004]. There is also an associated review article [http://openwetware.org/wiki/PMID:_15653319 ] 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. <br> |
Below you will find some questions to guide your reading of the Molecular Cell article. <b> You do not have to turn in the answers to these question. </b> 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. <br> | Below you will find some questions to guide your reading of the Molecular Cell article. <b> You do not have to turn in the answers to these question. </b> 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. <br> | ||
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* f. If the subject of the second paragraph was functional composition, what is the subject of the third paragraph? | * f. If the subject of the second paragraph was functional composition, what is the subject of the third paragraph? | ||
* g. The fourth paragraph begins to address the genetic and biochemical evidence for how the modules of SAGA fit together. Why is this transition/information included? | * g. The fourth paragraph begins to address the genetic and biochemical evidence for how the modules of SAGA fit together. Why is this transition/information included? | ||
− | * h. | + | * h. You ve been told over and over that yeast is a good model system for understanding more complex eukaryotic cells, like human, but what does the fifth paragraph suggest about that belief? Why do the authors spend time describing what s known about the human SAGA complex? <br> |
Next work on the <b>Results:</b><br> | Next work on the <b>Results:</b><br> | ||
− | *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. The following questions are intended to help you think about what | + | *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. The following questions are intended to help you think about what you re reading and do not require long answers. |
− | * Section 1: A 3d | + | * Section 1: A 3d model |
** a. Read [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10504710&query_hl=13&itool=pubmed_docsum| this abstract] and a little of the paper itself from the Nature Biotechnology link to get some idea of what TAP-tag purification is. | ** a. Read [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=AbstractPlus&list_uids=10504710&query_hl=13&itool=pubmed_docsum| this abstract] and a little of the paper itself from the Nature Biotechnology link to get some idea of what TAP-tag purification is. | ||
** b. Why was TAP-purification of SAGA from an ada1 deletion strain used as control for knowing that EM particles were actually SAGA? | ** b. Why was TAP-purification of SAGA from an ada1 deletion strain used as control for knowing that EM particles were actually SAGA? | ||
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** d. How many clefts? | ** d. How many clefts? | ||
* Section 2: Mapping Taf subcomplex | * Section 2: Mapping Taf subcomplex | ||
− | ** a. | + | ** a. Don t sweat the details of this section. Think more broadly about why a |