Difference between revisions of "20.109(S08):Initiate cell culture (Day2)"
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#The cells intended for your 2D culture probably do not need to be spun down again, but the cells for your 3D cultures must be directly resuspended in alginate rather than culture medium. While you spin down your alginate-bound cells, set up your 2D cultures according to the directions below. | #The cells intended for your 2D culture probably do not need to be spun down again, but the cells for your 3D cultures must be directly resuspended in alginate rather than culture medium. While you spin down your alginate-bound cells, set up your 2D cultures according to the directions below. | ||
#Label two T25 flasks with your group colour, initials, the date, and a description of the cells and culture conditions. Now make your cell dilution, using the example below as a guide - just change the volumes according to how many cells you want to add (always making 10 mL of culture total). | #Label two T25 flasks with your group colour, initials, the date, and a description of the cells and culture conditions. Now make your cell dilution, using the example below as a guide - just change the volumes according to how many cells you want to add (always making 10 mL of culture total). | ||
− | #* | + | #*Let s say you have 6M cells/mL in your conical tube, and want to use 600,000 cells to initiate your 2D cultures. Stand your T25 flasks upright and take the caps off. Start by adding 100 μL of chondrocytes against the back wall of each of your T25 flasks, making sure to touch only the sterile pipet tip to the flask, and to avoid touching the pipetman body inside this sterile area. (By back wall I mean what will become the bottom inside wall when you lay the flask down, which is the surface on which your cells grow.) |
#*Now add 9.9 mL of warm culture medium into each flask, and triterate to mix the cells. | #*Now add 9.9 mL of warm culture medium into each flask, and triterate to mix the cells. | ||
#*Finally, tighten the cap back onto the flask and put the cells in the incubator. | #*Finally, tighten the cap back onto the flask and put the cells in the incubator. | ||
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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 [ |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. | 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 [ |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. | ||
− | 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 |