Difference between revisions of "20.109(F08): Mod 2 Day 2 Yeast transformation"
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==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 | + | 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. | #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. | #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. 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 | + | #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 | + | #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. | #*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. | #*work on the Materials and Methods section of your upcoming lab report. | ||
− | #*periodically "flick" your tubes that are incubating at | + | #*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 |