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| [[Skatebro:Project08]]
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| ==Tips==
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| *Want about 50μL volume in each tube, 1 ng template DNA, 300nM of each primer (~200ng)
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| *Try running at 55c regardless of melt temps from primer design programs
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| *If no binding on first run, try a temp gradient trial from 48-64c
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| *Do a pcr clean up (elute down with 30 microL instead of more) and run a gel with control fragment similar in size
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| *Try running PCR reaction with betane for low GC sequences
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| ==Steps==
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| #Dilute miniprep product/plasmid DNA/template to a concentration of 1ng/μL, and label this tube 'XXXX for PCR'
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| #*Dilution review: If starting concentration of plasmid DNA = 50ng/μL, add to a tube 49μL of water and 1μL of the 50ng/μL plasmid DNA.
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| #Also prepare 25μM tubes of each primer (if you don't have them already)
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| #Add in order: (total reaction volume of 50μL)
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| #*49 μL of Tom's PCR mix
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| #*1ng template DNA
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| #*.6μL of each primer (forward and reverse) (.6 μL of the 25μM primer yields roughly 300 nM final concentration of that primer, which is the goal)
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| #Run the tubes on the thermocycler at something like: 95deg for 3:00, then cycle through: (a) 94deg for :30 (b) 55deg for :30 (c)68deg for 2:15, then 72deg for 10:00
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