»  PCR – Ready-to-go Beads

PCR – Ready-to-go Beads

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.