Difference between revisions of "Standard PCR"
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| − | + | Gradient/ Standard PCR | |
| + | 1. Create standard PCR mix. | ||
| + | a. 10ul 2X GoTaq Green PCR mix | ||
| + | b. 1ul forward primer (10mMl) | ||
| + | c. 1ul reverse primer (10mM) | ||
| + | d. 7ul dH2O | ||
| + | 19ul Total | ||
| + | Add 1ul of template DNA | ||
| + | 2. Using the table below, program the gradient (Δ°C) to match the desired temperature range from the standard temperature. ± values are approximate when indicated with ~. | ||
| − | + | Δ°C ±°C from Standard Temperature | |
| + | Δ12°C ±5°C | ||
| + | Δ14°C ~±6°C | ||
| + | Δ17°C ~±7°C | ||
| + | Δ19°C ~±8°C | ||
| + | Δ21°C ~±9°C | ||
| + | Δ24°C ±10°C | ||
| + | Δ27°C ~±11°C | ||
| + | Δ29°C ~±12°C | ||
| + | Δ30°C ±12.5°C | ||
| − | + | Thermal Profile | |
| − | + | • 1 cycle of 94°C for 4min | |
| − | + | • 30 cycles | |
| − | + | o 94°C for 15s | |
| − | + | o Annealing temperature/Gradient (Δ°C) for 15s | |
| − | + | o 74°C for 30s | |
| − | + | • 1 cycle of 74°C for 4min | |
| − | + | (The annealing temperature should be about 5°C below the lower of the Tm values of the 2 primers) | |
| − | + | This page was last modified on June 1, 2015, at 16:10. | |
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| − | annealing | ||
Revision as of 13:17, 2 June 2015
Gradient/ Standard PCR
1. Create standard PCR mix. a. 10ul 2X GoTaq Green PCR mix b. 1ul forward primer (10mMl) c. 1ul reverse primer (10mM) d. 7ul dH2O 19ul Total
Add 1ul of template DNA
2. Using the table below, program the gradient (Δ°C) to match the desired temperature range from the standard temperature. ± values are approximate when indicated with ~.
Δ°C ±°C from Standard Temperature
Δ12°C ±5°C Δ14°C ~±6°C Δ17°C ~±7°C Δ19°C ~±8°C Δ21°C ~±9°C Δ24°C ±10°C Δ27°C ~±11°C Δ29°C ~±12°C Δ30°C ±12.5°C
Thermal Profile • 1 cycle of 94°C for 4min • 30 cycles o 94°C for 15s o Annealing temperature/Gradient (Δ°C) for 15s o 74°C for 30s • 1 cycle of 74°C for 4min (The annealing temperature should be about 5°C below the lower of the Tm values of the 2 primers) This page was last modified on June 1, 2015, at 16:10.
