Smolke:Protocols/Library transformation: Difference between revisions
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(New page: {{Smolke_Top}} Still a work in progress ==Overview== Constructing large mutant libraries in ''S. cerevisiae'' by gap repair. ==Materials== *Mutazyme (Stratagene 200550) *2mm gap electro...) |
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==Overview== | ==Overview== | ||
Constructing large mutant libraries in ''S. cerevisiae'' by gap repair. | Constructing large mutant libraries in ''S. cerevisiae'' by gap repair. | ||
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#Clean up PCR product | #Clean up PCR product | ||
#*Commercial kits work just fine here | #*Commercial kits work just fine here | ||
#Using PCR product as template, set up 8x 100uL PCRs using | #Using PCR product as template, set up 8x 100uL PCRs using Pfu as the polymerase. | ||
#*For a 2.5kb template, reamplification with Taq introduced roughly 0.6 nucleotide mutations per template. Switching to Pfu made this step effectively faithful. | |||
#Combine all 800uL of PCR product (generally 40-50μg of DNA) into one tube. | #Combine all 800uL of PCR product (generally 40-50μg of DNA) into one tube. | ||
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#Run a gel to check that your PCR and digest worked | #Run a gel to check that your PCR and digest worked | ||
#Assuming success, make three tubes of DNA: | #Assuming success, make three tubes of DNA: | ||
## | ##375uL insert + 150uL vector | ||
## | ##375uL insert + 150uL vector | ||
## | ##450uL water + 75uL vector (Negative control) | ||
#Phenol-chloroform extract and ethanol precipitate tubes (can't use commercial columns since it's so much DNA) | #Phenol-chloroform extract and ethanol precipitate tubes (can't use commercial columns since it's so much DNA) | ||
#*DO NOT resuspend - you'll use the cell suspension to resuspend the DNA later. Just leave the DNA precipitated in the tube | #*DO NOT resuspend - you'll use the cell suspension to resuspend the DNA later. Just leave the DNA precipitated in the tube | ||
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#In the morning, dilute back to 50mL YPD at OD 0.1 | #In the morning, dilute back to 50mL YPD at OD 0.1 | ||
#Let the culture regrow to OD 1.3-1.5 (about 7 hours for me) | #Let the culture regrow to OD 1.3-1.5 (about 7 hours for me) | ||
#*Meanwhile, if you have frozen Tris-DTT, thaw it on ice about an hour before you need it. | |||
#*Also, chill your buffer E and the tubes holding the precipitated DNA | |||
#Add 500uL Tris-DTT, return culture to incubator for 10-15 minutes | #Add 500uL Tris-DTT, return culture to incubator for 10-15 minutes | ||
#* | #*Place an aliquot of YPD in the incubator now (to warm it for the transformations) | ||
#Spin down at 2500g, 4°C, 3min | #Spin down at 2500g, 4°C, 3min | ||
#Wash with 25mL ice cold buffer E, then 1mL buffer E (decant supernatant, resuspend, respin). | #Wash with 25mL ice cold buffer E, then 1mL buffer E (decant supernatant, resuspend, respin). |
Latest revision as of 14:05, 20 October 2009
OverviewConstructing large mutant libraries in S. cerevisiae by gap repair. Materials
ProcedureDNA PrepBest done the day before you plan to do your transformations. The backbone digest should run overnight (finishing the morning of the transformations). You can run the second round of PCRs on the day of the transformation, but I like to do them overnight as well (so they're ready the morning of). Insert
Backbone
Coprecipitation
Transformation
Notes
ReferencesFor the original (more complete and detailed) description of the protocol:
Contact |