Difference between revisions of "Standard PCR"

From GcatWiki
Jump to: navigation, search
 
Line 1: Line 1:
 
   Gradient/ Standard PCR
 
   Gradient/ Standard PCR
1. Create standard PCR mix.
+
1.Create standard PCR mix.
a. 10ul 2X GoTaq Green PCR mix
+
 
b. 1ul forward primer (10mMl)
+
10ul 2X GoTaq Green PCR mix
c. 1ul reverse primer (10mM)
+
 
d. 7ul dH2O
+
1ul forward primer (10mMl)
 +
 
 +
1ul reverse primer (10mM)
 +
 
 +
7ul dH2O
 +
 
 
19ul Total
 
19ul Total
              Add 1ul of template DNA
+
 
 +
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 ~.  
 
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
+
Δ°C         ±°C from Standard Temperature
Δ12°C ±5°C
+
Δ12°C     ±5°C
Δ14°C ~±6°C
+
 
Δ17°C ~±7°C
+
Δ14°C     ~±6°C
Δ19°C ~±8°C
+
 
Δ21°C ~±9°C
+
Δ17°C     ~±7°C
Δ24°C ±10°C
+
 
Δ27°C ~±11°C
+
Δ19°C     ~±8°C
Δ29°C ~±12°C
+
 
Δ30°C ±12.5°C
+
Δ21°C     ~±9°C
 +
 
 +
Δ24°C     ±10°C
 +
 
 +
Δ27°C     ~±11°C
 +
 
 +
Δ29°C     ~±12°C
 +
 
 +
Δ30°C     ±12.5°C
 +
 
 +
 
 +
  Thermal Profile
  
Thermal Profile
 
 
• 1 cycle of 94°C for 4min
 
• 1 cycle of 94°C for 4min
• 30 cycles
+
 
o 94°C for 15s
+
• 30 cycles of 94°C for 15s, Annealing temperature/Gradient (Δ°C) for 15s, 74°C for 30s
o Annealing temperature/Gradient (Δ°C) for 15s
+
 
o 74°C for 30s
 
 
• 1 cycle of 74°C for 4min
 
• 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)
 
(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.
 
This page was last modified on June 1, 2015, at 16:10.

Latest revision as of 11:31, 2 June 2015

  Gradient/ Standard PCR

1.Create standard PCR mix.

10ul 2X GoTaq Green PCR mix

1ul forward primer (10mMl)

1ul reverse primer (10mM)

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 of 94°C for 15s, Annealing temperature/Gradient (Δ°C) for 15s, 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.