Standard PCR Conditions

Prepare a Master Mix for appropriate Taq polymerase containing the following amounts of each component PER REACTION.  Make enough Master Mix for N+1 reactions. 

Master Mix:

*these amounts can change depending on the Taq used, so make sure you are following the correct concentrations for the correct Taq

Promega Taq DNA Polymerase

Component           Starting pointFor 20ul reation
 Buffer1x4ul of 5x
Primers0.5 pMol0.5ul of each of 20 pMol
dNTP1mM (.25mM of each dNTP)          0.5ul of 10mM
Taq5U/ul units0.12ul
Template

~50ng genomic

~10ng plasmid

Depends on your concentration        

 

If these conditions do not work, Mg is one of the first things to add, after trying a temperature gradient.  Do a gradient of 0.5mM increments.

 

Phusion DNA Polymerase (*Polymerase is in the Master mix)

Component         Starting point         For 20ul reation
Master mix1x10ul of 2x
Primers0.5 pMol0.5ul of each of 20 pMol
Template

~50ng genomic

~10ng plasmid

Depends on your concentration           

 

If these conditions do not work, DMSO is one of the first things to add, specifically for GC-rich amplicons, after trying a temperature gradient. Add in 0.6ul incriments.

 

Cycle Conditions for Taq Polymerase

 When you are first trying a PCR, it is often useful to do a temperature gradient.  Use the following guidelines for designing your program.  

ConditionsGuidelines
DenaturationTemp: 95°C. Time: 2 min on initial cycle; 30 seconds to 1 min on rest
AnnealingTemp: 5°C below Tm of primers; no lower than 40°C.  Time:  30-45 seconds.  This is the step where you would use a gradient.       
ExtensionTemp: 72°C.  Time:  ~1 min/kb of expected product; 5-10 min on last cycle.
Number of Cycles       ~30 cycles

 

Cycle Conditions for Taq Polymerase

 When you are first trying a PCR, it is often useful to do a temperature gradient.  Use the following guidelines for designing your program.  

ConditionsGuidelines
DenaturationTemp: 95°C. Time: 2 min on initial cycle; 30 seconds on rest
AnnealingTemp: 5°C below Tm of primers; no lower than 40°C.  Time:  30 seconds.  This is the step where you would use a gradient.       
ExtensionTemp: 72°C.  Time:  ~1 min/kb of expected product; 5 min on last cycle.
Number of Cycles         ~35 cycles

 

Cycle Conditions for Phusion Polymerase  

ConditionsGuidelines
DenaturationTemp: 98°C. Time: 30 sec on initial cycle; 10 seconds on rest
AnnealingTemp: 5°C below Tm of primers; no lower than 40°C.  Time: 20 seconds.  This is the step where you would use a gradient.        
ExtensionTemp: 72°C.  Time:  ~20 sec/kb of expected product; 5 min on last cycle.
Number of Cycles          ~35 cycles

 

Here is a sample PCR Program, using a wide gradient, for an expected product of about 1kb.  The first step of 95 forever is just to heat the block before you add your tubes, and you would then press "Edit" -> "Skip Step" to continue to step 2.  Step 8 is just to hold your PCR at a low temperature until you take it out.  Do not leave in overnight!

Step             

Temperature (°C)                Time (min)          

1

95infinite

2

952:00

3

950:30

4

45 – 650:30

5

721:00

 

Repeat 3-5 34x 

7

725:00

8

4infinite

 

Use Veriflex option for temperature gradient. You can select 2/3 temperatures across the PCR block, depending on the thermocycler you use.  

For specific instructions on how to enter your program into the thermocycler, see the manual for the thermocycler you want to use. Manuals can be found in Manter 335, or in the equipment manual folder in Box.