Skatebro:PCR: Difference between revisions
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==Steps== | ==Steps== | ||
#Dilute | #Dilute plasmid/template DNA to a concentration of 1ng/μL, and label this tube 'XXXX for PCR' | ||
#*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. | #*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. | ||
#Also prepare 25μM tubes of each primer (if you don't have them already) | #Also prepare 25μM tubes of each primer (if you don't have them already) | ||
Revision as of 20:32, 31 January 2008
Tips
- Want about 50μL volume in each tube, 1 ng template DNA, 300nM of each primer (~200ng)
- Try running at 55c regardless of melt temps from primer design programs
- If no binding on first run, try a temp gradient trial from 48-64c
- Do a pcr clean up (elute down with 30 microL instead of more) and run a gel with control fragment similar in size
- Try running PCR reaction with betane for low GC sequences
Steps
- Dilute plasmid/template DNA to a concentration of 1ng/μL, and label this tube 'XXXX for PCR'
- 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.
- Also prepare 25μM tubes of each primer (if you don't have them already)
- Add in order: (total reaction volume of 50μL)
- 49 μL of Tom's PCR mix
- 1ng template DNA
- .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)
- 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
