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Protocol Purifying DNA for Sequencing ***Change pipet tips EVERY time you pipet something*** Summary: We will be sequencing the DNA sequences of the three virus genes you amplified using pcr, but first, we need to clean up the DNA. Pcr reactions contain DNA, primers, buffer, MgCl2, taq polymerase, and dNTPs. We must remove everything except for the DNA to get it sequenced. 1) Label 3 microtubes tubes with 500 µl-capacity: psbA (virus #) DNApol (virus #) g23 (virus #) 2) Add 200 µl of Buffer PB to each tube (you can use the same pipet tip): 200 µl 200 µl Buffer PB 200 µl psbA virus DNApol virus g23 virus 3) Get your pcr products from your viruses (you do NOT need your positive or negative control) 4) Add 40 µl of your pcr products to the appropriate tubes with the buffer **this will use ALL of your pcr product** psbA pcr product DNApol pcr product g23 pcr product 40 µl 40 µl 40 µl psbA virus DNApol virus g23 virus 5) Label 3 microtubes tubes with 2 ml-capacity; **label ON THE SIDE of the tube** psbA (virus #) DNApol (virus #) g23 (virus #) 6) Put MinElute columns into the 3 labeled microtubes with 2 ml-capacity: psbA (virus #) DNApol (virus #) g23 (virus #) 7) Pipet all of your samples mixed with Buffer PB into the MinElute columns: psbA virus DNApol virus g23 virus 240 µl psbA virus 240 µl DNApol virus 240 µl g23 virus 8) Centrifuge MinElute columns and tubes at 13000 rpm for 1 minute. 9) Pour out the liquid that went through the column into a waste beaker. -KEEP the column and the tube 10) Add 750 µl of Buffer PE to each MinElute column: psbA virus µl 750 750 µl Buffer PE 75 DNApol virus 0µ l g23 virus 11) Centrifuge MinElute columns and tubes at 13000 rpm for 1 minute. 12) Pour out the liquid that went through the column into a waste beaker. -KEEP the column and the tube 13) Label 3 microtubes tubes with 1.5 ml-capacity; **label ON THE SIDE of the tube** psbA (virus #) DNApol (virus #) g23 (virus #) 14) Transfer your MinElute columns into the newly-labeled microtubes with 1.5 ml-capacity 15) Add 10 µl of Buffer EB to each MinElute column - put the buffer DIRECTLY onto the column membrane: psbA virus 10 µl 10 µl Buffer EB 10 DNApol virus µl g23 virus 16) Let the columns sit for 1 minute. 17) Centrifuge MinElute columns and tubes at 13000 rpm for 1 minute. 18) KEEP THE LIQUID THAT WENT THROUGH THE MINELUTE COLUMN -This is your DNA!!! 19) Discard the MinElute Columns and close your tubes. 19) Put your recovered DNA into the ice bucket.