Big Dye Sequencing Protocol

Reaction Mixes

20 µl Reaction:

• 4 µl Big Dye Mix
  
• 4 µl 5X CSA buffer
  
• 3-4 pmoles primer
  
• 200-500 ng template
  
• 1-2 µl DMSO
  
• H2O to final volume of 20 µl
  

10 µl Reaction:

• 4 µl Big Dye Mix
  
• 3-4 pmoles primer
  
• 200-500 ng template
  
• 0.5-1 µl DMSO
  
• H2O to final volume of 10 µl
  

Note #1: 5X CSA buffer is a PE Applied Biosystems product. It is located in the Biotech lab where the sequencing reagents are kept. It should be kept on ice.

Note #2: DMSO is optional, but will not hurt the reaction.

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PCR Cycle (for PE 9600 or equivalent)

• 95° C for 5 minutes
  

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• 95° C for 10 seconds
  
• 50° C for 5 seconds
  
• 60° C for 4 minutes 
  
• µ==> 25 cycles
  

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• 4° C hold
  

Note #3: Ramping time is very important; should not be slower than 1 degree/second.