Smolke:Protocols/Chromosomal Modification: Difference between revisions
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*Anecdotally, I had about half of my first round of colonies (Step 3, above) integrate into the correct locus. A strain with integration into the wrong locus cannot recombine out the URA marker. I was integrating a fluorescent protein, so identification of the correct locus was straightforward - cells with the correct integration showed similar fluorescence histograms. Cells with the wrong locus showed a different mean fluorescence. | *Anecdotally, I had about half of my first round of colonies (Step 3, above) integrate into the correct locus. A strain with integration into the wrong locus cannot recombine out the URA marker. I was integrating a fluorescent protein, so identification of the correct locus was straightforward - cells with the correct integration showed similar fluorescence histograms. Cells with the wrong locus showed a different mean fluorescence. | ||
*If you're integrating a fluorescent protein, checking for integration is easy - just scrape cells off a plate, resuspend in PBS, and run on the flow cytometer. | *If you're integrating a fluorescent protein, checking for integration is easy - just scrape cells off a plate, resuspend in PBS, and run on the flow cytometer. | ||
*After removing the marker (step 9 above), <10% of the resulting cells still contained the integration cassette. The rest had just recombined out the original plasmid (recombination through YIp-In rather than the desired YIp-Out). As mentioned, checking for fluorescent cassettes is easy. Otherwise, screen by colony PCR or check for loss of the wt integration locus (so look for lys- colonies if using pCS1441). | |||
*Again, anecdotally, integration into the lys2 locus (pCS1441) gave similar mean fluorescence to a cen4 centromeric plasmid (pCS2), but a much reduced variation in expression (CV decreased by ~2x). | *Again, anecdotally, integration into the lys2 locus (pCS1441) gave similar mean fluorescence to a cen4 centromeric plasmid (pCS2), but a much reduced variation in expression (CV decreased by ~2x). | ||
Revision as of 11:46, 22 October 2009
KnockoutsTalk to Mike. InsertionsPCR MethodTalk to Mike. Plasmid MethodOverviewWe use the disintegrator vectors[1] from EUROSCARF, stored as pCS1439 to 1441. Protocol
Notes
References |