Standard PCR - Revision history

Eckdahl at 13:31, 2 June 2015

2015-06-02T13:31:22Z

Eckdahl at 13:17, 2 June 2015

2015-06-02T13:17:41Z

Eckdahl at 13:51, 1 October 2011

2011-10-01T13:51:59Z

XiZhu at 19:06, 9 June 2008

2008-06-09T19:06:40Z

XiZhu at 19:05, 9 June 2008

2008-06-09T19:05:30Z

New page

A. Reaction Components:

2 ul 10X PCR buffer w/ 15 mM MgCl2

2 ul 2 mM dNTPs (working concentration 2 mM each)

1 ul forward primer (20 pmol)

1 ul reverse primer (20 pmol)

1 ul Taq DNA polymerase

12 ul dH2O

19 ul total

add 1 ul template DNA

Creat masta 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.

@@ Line 1: / Line 1: @@
     Gradient/ Standard PCR
-.	Create standard PCR mix.
+.Create standard PCR mix.
-a.	10ul 2X GoTaq Green PCR mix
-b.	1ul forward primer (10mMl)
+ul 2X GoTaq Green PCR mix
-c.	1ul reverse primer (10mM)
-d.	7ul dH2O
+ul forward primer (10mMl)
 ul Total
-              Add 1ul of template DNA
 .	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
 •	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
 •	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.

@@ Line 1: / Line 1: @@
-    A. Reaction Components:
+    Gradient/ Standard PCR
-ul 10X PCR buffer w/ 15 mM MgCl2
+  Δ°C	±°C from Standard Temperature
-ul 2 mM dNTPs (working concentration 2 mM each)
+Thermal Profile
+•	1 cycle of 94°C for 4min
-ul forward primer (20 pmol)
+•	30 cycles
+o	94°C for 15s
-ul reverse primer (20 pmol)
+o	Annealing temperature/Gradient (Δ°C) for 15s
+o	74°C for 30s
-ul Taq DNA polymerase
+•	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)
-ul dH2O
+This page was last modified on June 1, 2015, at 16:10.

@@ Line 17: / Line 17: @@
 add 1 ul template DNA
-Creat masta mix if desired
+Create master mix if desired
     B. Thermal Profile