» 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 (10M)
Seal with caps or wax.
Thermal Profiles:
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Basic program |
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94 C 5:00 |
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94 C :30 |
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50 C :30 |
can increase the length of annealing up to 1 minute |
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72 C :45 |
can increase the length of extension up to 1:30 (for longer genes). |
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35 cycles |
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72 C 7:00 |
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hold at 10 C |
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Rock and Roll |
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94 C 5:00 |
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94 C :15 |
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42 C :05 |
can increase the length of annealing up to :30 |
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68 C :15 |
can increase the length of annealing up to :30 |
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40 cycles |
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72 C for 7:00 |
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hold at 10 C |
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Down/Up |
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94 C 5:00 |
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94 C :30 |
94 C :30 |
94 C :30 |
94 C :30 |
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54 C :45 |
50 C :45 |
52 C :45 |
54 C :45 |
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72 C 1:30 |
72 C 1:30 |
72 C 1:30 |
72 C 1:30 |
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10 cycles |
10 cycles |
10 cycles |
10 cycles |
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72 C 7:00 |
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hold at 10 C |
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This can be useful for double bands, and you can use any range of annealing temperatures. |
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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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