Difference between revisions of "PCR 2X Master Mix"

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(Created page with "The master mix can be purchased premixed commercially or mixed from the individual components. For a single 25 µl reaction :2.5 µl 10X buffer (usually this is supplied wit...")
 
 
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For a single 25 µl reaction
 
For a single 25 µl reaction
:2.5 µl 10X buffer (usually this is supplied with the polymerase, this assumes it does not already have  
+
:2.5 µl 10X [[Taq Buffer]] (usually this is supplied with the polymerase, this assumes it does not already have MgCl<sub>2</sub>.)
 
:0.5 µl 10mM dNTPs
 
:0.5 µl 10mM dNTPs
 
:1 µl 50mM MgCl<sub>2</sub> (adjusting the Mg concentration affects the specificity of the polymerase and can be optimized)
 
:1 µl 50mM MgCl<sub>2</sub> (adjusting the Mg concentration affects the specificity of the polymerase and can be optimized)
:0.5 µl (5 U / µl) Taq polymerase
+
:0.5 µl (5 U / µl) thermostable (Taq) polymerase
:8 µl nuclease-free H<sub>2</sub>O (adjust if necessary if MgCl<sub>2</sub> or Taq volume is adjusted for a 12.5 µl total volume.  
+
:8 µl nuclease-free H<sub>2</sub>O (adjust if necessary if MgCl<sub>2</sub> or Taq volume is adjusted for a 12.5 µl total volume).  
  
 
In general a mix would be made for many reactions, say for 100, and the volumes multiplied accordingly. The mix can be stored at -20 C.
 
In general a mix would be made for many reactions, say for 100, and the volumes multiplied accordingly. The mix can be stored at -20 C.
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 +
=What Links Here=
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Latest revision as of 23:59, 16 July 2018

The master mix can be purchased premixed commercially or mixed from the individual components.

For a single 25 µl reaction

2.5 µl 10X Taq Buffer (usually this is supplied with the polymerase, this assumes it does not already have MgCl2.)
0.5 µl 10mM dNTPs
1 µl 50mM MgCl2 (adjusting the Mg concentration affects the specificity of the polymerase and can be optimized)
0.5 µl (5 U / µl) thermostable (Taq) polymerase
8 µl nuclease-free H2O (adjust if necessary if MgCl2 or Taq volume is adjusted for a 12.5 µl total volume).

In general a mix would be made for many reactions, say for 100, and the volumes multiplied accordingly. The mix can be stored at -20 C.

What Links Here