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PCR – Ready-to-go Beads

Recipe: 2uL DNA
21uL H2O
1uL each direction of primer (10M)

Seal with caps or wax.

Thermal Profiles:

Basic program
94 C 5:00  
94 C :30  
50 C :30 can increase the length of annealing up to 1 minute
72 C :45 can increase the length of extension up to 1:30 (for longer genes).
35 cycles
72 C 7:00  
hold at 10 C  

 

Rock and Roll
94 C 5:00  
94 C :15  
42 C :05 can increase the length of annealing up to :30
68 C :15 can increase the length of annealing up to :30
40 cycles
72 C for 7:00  
hold at 10 C  

 

Down/Up
94 C 5:00      
94 C :30 94 C :30 94 C :30 94 C :30
54 C :45 50 C :45 52 C :45 54 C :45
72 C 1:30 72 C 1:30 72 C 1:30 72 C 1:30
10 cycles 10 cycles 10 cycles 10 cycles
72 C 7:00      
hold at 10 C      
This can be useful for double bands, and you can use any range of annealing temperatures.

Recommendations: 
12s: basic program, 50 C or 48 C, go as low as 46 C
16s: basic program, 50 C or 48 C
18s: basic program, 50 C or 48 C
28s: basic program, 50 C or 48 C
COI: Rock and roll, start at 48 C, go as low as 42 C
ExtA/B: Rock and roll with longer annealing/extension, start at 48 C, usually best at 42 C
Histone3: basic program, 50 C or 52 C
EF-1 alpha: basic program with longer annealing and extension, 54 C or 56 C or Down/Up.

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