Endy:Library-based construction/Inverter library protocol: Difference between revisions
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'''We should be fleshing this out as we work through it a few times''' | '''We should be fleshing this out as we work through it a few times''' | ||
==Generate inverter library via mutagenic PCR== | |||
*50 ul PCR reaction using Stratagene's [http://www.stratagene.com/products/displayProduct.aspx?pid=614 GeneMorph® II Random Mutagenesis kit] | |||
*PCR cleanup and elute with 30 ul | |||
==Digest the library and ligate into [[Endy:Screening plasmid|SP1.0]]== | |||
*Example: 50 ul [[Knight:Restriction Digest|restriction digest]] using all of the PCR reaction | |||
**Separate the insert out on a gel and perform gel extraction | |||
**Elute the entire reaction into a single 30 ul volume (reuse on multiple columns if necessary) | |||
**Perform two 20-ul ligations with 15 ul of the digest in each and 1 ul of the cut SP1.0 vector | |||
***Can also save some of the ligation and use less, probably don't need this much | |||
==Transform into electrocompetent CW2553/pJat8== | |||
#Grow for one hour without antibiotic (1ml), then move into a higher volume (50ml) of selective media | #Grow for one hour without antibiotic (1ml), then move into a higher volume (50ml) of selective media | ||
#*plate a portion on selecive media in order to determine the libary size. | #*plate a portion on selecive media in order to determine the libary size. | ||
#Store 5 glycerols of the libary from the overnight culture | #Store 5 glycerols of the libary from the overnight culture | ||
#Start experimental cultures with no induction and high induction | #Start experimental cultures with no induction and high induction | ||
#Sort cells with LOW in/HIGH out and HIGH in/LOW out into sub-libraries | #Sort cells with LOW in/HIGH out and HIGH in/LOW out into sub-libraries | ||
#*Be sure to also collect analytical samples of the populations after sorting, so that we have data of what the libraries looked like. | #*Be sure to also collect analytical samples of the populations after sorting, so that we have data of what the libraries looked like. | ||
#Grow up the sub-libraries under the reverse condition (and the same condition) | #Grow up the sub-libraries under the reverse condition (and the same condition) | ||
#*The reason for growing under the same conditions is to see how effective the sorting was (control, basically). | #*The reason for growing under the same conditions is to see how effective the sorting was (control, basically). | ||
#Conduct second round of sorting to create sub-libraries that we're tested under both HIGH input and LOW input conditions | #Conduct second round of sorting to create sub-libraries that we're tested under both HIGH input and LOW input conditions | ||
#Grow up library and store 5 glycerols, as well as plate to get individual mutants | #Grow up library and store 5 glycerols, as well as plate to get individual mutants | ||
#Test individual mutants, if necesary can repeat the rounds of screening to tighten up the library | #Test individual mutants, if necesary can repeat the rounds of screening to tighten up the library | ||
Revision as of 12:15, 18 July 2006
We should be fleshing this out as we work through it a few times
Generate inverter library via mutagenic PCR
- 50 ul PCR reaction using Stratagene's GeneMorph® II Random Mutagenesis kit
- PCR cleanup and elute with 30 ul
Digest the library and ligate into SP1.0
- Example: 50 ul restriction digest using all of the PCR reaction
- Separate the insert out on a gel and perform gel extraction
- Elute the entire reaction into a single 30 ul volume (reuse on multiple columns if necessary)
- Perform two 20-ul ligations with 15 ul of the digest in each and 1 ul of the cut SP1.0 vector
- Can also save some of the ligation and use less, probably don't need this much
Transform into electrocompetent CW2553/pJat8
- Grow for one hour without antibiotic (1ml), then move into a higher volume (50ml) of selective media
- plate a portion on selecive media in order to determine the libary size.
- Store 5 glycerols of the libary from the overnight culture
- Start experimental cultures with no induction and high induction
- Sort cells with LOW in/HIGH out and HIGH in/LOW out into sub-libraries
- Be sure to also collect analytical samples of the populations after sorting, so that we have data of what the libraries looked like.
- Grow up the sub-libraries under the reverse condition (and the same condition)
- The reason for growing under the same conditions is to see how effective the sorting was (control, basically).
- Conduct second round of sorting to create sub-libraries that we're tested under both HIGH input and LOW input conditions
- Grow up library and store 5 glycerols, as well as plate to get individual mutants
- Test individual mutants, if necesary can repeat the rounds of screening to tighten up the library
