Difference between revisions of "Standard PCR"

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   A. Reaction Components:
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   Gradient/ Standard PCR
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1. Create standard PCR mix.
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a. 10ul 2X GoTaq Green PCR mix
 +
b. 1ul forward primer (10mMl)
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c. 1ul reverse primer (10mM)
 +
d. 7ul dH2O
 +
19ul Total
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              Add 1ul of template DNA
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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 ~.  
  
2 ul 10X PCR buffer w/ 15 mM MgCl2
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  Δ°C ±°C from Standard Temperature
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Δ12°C ±5°C
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Δ14°C ~±6°C
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Δ17°C ~±7°C
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Δ19°C ~±8°C
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Δ21°C ~±9°C
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Δ24°C ±10°C
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Δ27°C ~±11°C
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Δ29°C ~±12°C
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Δ30°C ±12.5°C
  
2 ul 2 mM dNTPs (working concentration 2 mM each)
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Thermal Profile
 
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1 cycle of 94°C for 4min
1 ul forward primer (20 pmol)
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• 30 cycles
 
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o 94°C for 15s
1 ul reverse primer (20 pmol)
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o Annealing temperature/Gradient (Δ°C) for 15s
 
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o 74°C for 30s
1 ul Taq DNA polymerase
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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)
12 ul dH2O
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This page was last modified on June 1, 2015, at 16:10.
 
 
19 ul total
 
 
 
add 1 ul template DNA
 
 
 
Create master mix if desired
 
 
 
  B. Thermal Profile
 
 
 
1 circle of : 94 C 4 mins
 
 
 
30 circles of:  94 C 15 sec.; annealing temp. 15 sec.; 74 C 30 sec.
 
 
 
1 circle of : 74 C 4 mins
 
 
 
annealing temp. should be about 4 C below the lower of the Tm values for the two primers.
 

Revision as of 11:17, 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.