(3) Which plates show evidence of the lacZ gene having been cut by Cas9 (1 pt)?
Added by Derrick J.
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This typically involves a plasmid containing the lacZ gene and a guide RNA designed to direct Cas9 to the specific location within the lacZ gene. Show more…
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B: Which plates show evidence of the lacZ gene having been cut by Cas9? Plate C: Of the plates that show evidence of the lacZ gene having been cut, which also show evidence of the DNA cut having been repaired? Note that repairing DNA does not mean repairing the function of a gene: Plate
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You are attempting to knockout the gene pictured below. The gene has 3 exons, and the enzymatic activity of the protein is within exon 2. You design a traditional gene targeting strategy so that the entire 2nd exon and a part of the following intron up to and including the Hind III site pictured is deleted, and replaced with a NeoR cassette. You introduce the targeting construct into ES cells. 1) Using the 5' probe noted in the picture, you set up a Southern blot using genomic DNA from ES cells digested with Hind III to screen your ES cells that survived drug treatment. Assume that the size of the fragment that recombines into the genomic DNA is roughly the same size as the endogenous piece that it replaces. If the targeting of 1 allele is successful, what size band(s) would you expect to see? 2) Draw and fully label the complete genomic region of the successfully targeted ES cell (described in question #1) below (hint: mice are diploid). 3) When screening your ES cells you forget to add Gangciclovir. What would be the consequence of your error? 4) If you want to study a mouse where this gene is only targeted in intestinal cells, which transgenic mouse would you cross your conditional knock-out mouse to?
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Aval (C^PyCGPuG (Py stands for pyrimidine, Pu for purine) Sal I (G^TCGAC) TaqI (T^CGA) XhoI (C^TCGAG) Above is a list of restriction enzymes and their recognition sequences. (^) indicates where each enzyme cuts. A plasmid is cut with Sal I, and combined with genomic DNA digested with XhoI. In those plasmids with a genomic fragment, the Sal I- XhoI junction can be cut with: SalI only, XhoI only, both original enzymes, either Sal I or TaqI, TaqI only. If a PCR amplified sample hybridizes with two allele specific probes, one normal and one mutant sequence, a carrier sequence is identified if: a. Both probes give a signal b. Neither sample gives a signal c. Only the mutant probe gives a signal d. Only the non-mutant probe gives a signal e. None of the choices is correct
Supreeta N.
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