Skatebro:PCR

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(Steps)
Current revision (06:06, 9 February 2008) (view source)
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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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