In week 13 you cloned the coding sequence (start codon to stop codon) or promoter fragment of a gene from corn into a Gateway entry vector (pENTR/SD/D-TOPO). Describe the steps that you would perform to clone a human gene from genomic DNA into the same gateway entry vector to make an entry clone (assume the gene has just one exon) (describe the main steps and components e.g. enzymes, primers, checks, etc. that you would use). The starting point is you and the end point is a confirmed entry clone for the gene.
Added by Xavier C.
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g., blood, tissue). - Use a DNA extraction kit or standard phenol-chloroform extraction method to isolate genomic DNA from the cells. Show more…
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Briefly describe the main steps required to clone a gene and produce a protein
Adi S.
6. NOTE: this will require that you remember some stuff from bacterial genetics. One thing that will help you, though, is the idea of a polycistronic mRNA. We'll talk about this more in the last unit of the course, but here you can have multiple distinct translated regions within one piece of mRNA downstream of just one promoter. So you'll effectively make a piece of mRNA with multiple start and stop codons, such that multiple, distinct proteins can be made from this one piece of mRNA. OK. So here we go. Our potential vector (depicted below) codes for one of these polycistronic mRNAs. a) You want to research the eukaryotic protein BabyFeet, which smells like peppermint. To make cDNA you need... b) You prepare plates to grow your bacteria on. Your competent E. coli are resistant to penicillin, cephalexin, and susceptible to ampicillin. Which antibiotic will you put in your media? c) Next, you prepare your cloning vector, which contains the genes Ampr, cepr, and penr all downstream of the promoter; all of these genes confer resistance to that particular antibiotic. The plasmid map below shows you the locations of many restriction sites, indicated by the restriction enzyme (x______) that cuts at that spot. Assume these restriction enzymes will all work for your cDNA as well. Which restriction enzyme would be best for your experiment? Why? d) Describe how you will get your donor into your plasmid. e) You isolate whole plasmids by gel electrophoresis. Then you transform your E. coli with the plasmid. Finally you plate E. coli onto the media you prepared with ampicillin (amp). How will you know if your vector was successfully incorporated into the genome?
Shaiju T.
You have cloned the Pepsi gene into the vector pKEN. You used two restriction enzymes, BamHI and EcoRI, to facilitate the cloning in a directional manner. The plasmid (pKEN) is approximately 5 kb long, and the insert Pepsi is 800 bp long. After transforming into E. coli, you grow multiple colonies and isolate the vector. Upon digesting the vector, it appears that there are three potential clones. To ensure that you have the correct clone, you need to sequence the gene. However, you must sequence both strands of the entire gene, and the only primers you have are for the end of the Pepsi gene. Please describe how you would sequence the entire gene, including the ends of the gene.
Sri K.
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