Difference between revisions of "20.109(S10):Initiate cell culture (Day2)"
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Did you know that NCBI has a whole site devoted to [http://www.ncbi.nlm.nih.gov/projects/genome/guide/cow/ all things cow]? | Did you know that NCBI has a whole site devoted to [http://www.ncbi.nlm.nih.gov/projects/genome/guide/cow/ all things cow]? | ||
− | + | It s true! And today you will use this site to find the primers you need to perform RT-PCR on Day 4 of this module. Try searching for collagen types I and II (the alpha chain of each is fine) in the '''Map Viewer''' (upper right of page). What chromosomee is each collagen chain located on? See if you can make your way to the UniSTS entries for collagen, which list recommended primers for RT-PCR. How long are the expected PCR products if these primers are used? | |
Another option for finding primer suggestions is looking in the literature. Of course, this can be a risky proposition, but if you verify the primers against information in the NCBI database, it can be faster than making your own from scratch, and provide a feeling of security (someone, somewhere has succesfully amplified the sequence in question!). The paper by [http://www.ncbi.nlm.nih.gov/pubmed/12507587 Ikenooue et al.] lists primers recommended for collagen type II. What species are the primers for? If it's not bovine, you cannot use the primers directly. However, you can BLAST the primers against the bovine genome, similar to what you did in Module 2 to verify your mutagenized plasmids against the original, or Module 1 to search for homology in your RNA aptamers. | Another option for finding primer suggestions is looking in the literature. Of course, this can be a risky proposition, but if you verify the primers against information in the NCBI database, it can be faster than making your own from scratch, and provide a feeling of security (someone, somewhere has succesfully amplified the sequence in question!). The paper by [http://www.ncbi.nlm.nih.gov/pubmed/12507587 Ikenooue et al.] lists primers recommended for collagen type II. What species are the primers for? If it's not bovine, you cannot use the primers directly. However, you can BLAST the primers against the bovine genome, similar to what you did in Module 2 to verify your mutagenized plasmids against the original, or Module 1 to search for homology in your RNA aptamers. | ||
− | Go to the [http://www.ncbi.nlm.nih.gov/blast/Blast.cgi BLAST site] and select the ''bos taurus'' genome. Type in the primers from the journal article one at a time, then perform the BLAST as follows: select BLASTN, change the | + | Go to the [http://www.ncbi.nlm.nih.gov/blast/Blast.cgi BLAST site] and select the ''bos taurus'' genome. Type in the primers from the journal article one at a time, then perform the BLAST as follows: select BLASTN, change the |