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| [[Skatebro:Project08]]
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| ==Tips==
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| *50μL volume total volume
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| *1 ng template DNA and [300nM] of each primer (~200ng)
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| *Run at 55c regardless of melt temps from primer design programs
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| **2nd pass: run a temp gradient trial from 48-64c
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| **3rd pass: run PCR reaction with betane for low GC sequences
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| *Do a pcr clean up (elute with 30μL) and run a gel
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| ==Controls==
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| *Digest a control fragment of similar length and also run on gel
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| ==Steps==
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| #Dilute plasmid/template DNA to a concentration of 1ng/μL, and label this tube 'XXXX for PCR'
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| #*Dilution review: If start with 50ng/μL plasmid DNA, add to a tube 49μL of water and 1μL of the 50ng/μL plasmid DNA.
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| #Prepare 25μM tubes of each primer (if you don't have them already)
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| #Add in order: (50μL in little pcr-sized tube)
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| #*49μL of Tom's PCR mix
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| #*1μL template DNA (=1 ng template DNA)
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| #*.6μL of each primer (forward and reverse) (.6 μL of the 25μM primer yields roughly [300 nM])
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| #Run the tubes on the thermocycler at an appropriately designed program:
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| #*~95deg for 3:00
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| #*30 cycles of (a) melt (b) anneal (c) extend
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| #*~72deg for 10:00
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