Standard PCR Conditions

Promega Taq DNA Polymerase

 

Component

Suggested Range

Starting point

For 20ul reation

10x Buffer

1x

1x

2ul of 10x

MgCl2*

0.5 – 9.0mM

1.5mM

1.2ul of 25mM

Primers

0.5 pMol

0.5 pMol

0.5ul of 20 pMol

dNTP

10-500mM

10mM

2.5ul of 10mM

Taq

1-2.5 units

2 units

0.5ul

Template

105 - 106

~50ng genomic

~10ng plasmid

Depends on your concentration

 

* Check to make sure that this isnŐt already in the buffer – if it is, donŐt add separately!  Mg is one of the first things to change if your PCR does not work, after trying a temperature gradient.  Do a gradient of 0.5mM increments.

 

Cycle Conditions

 

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

 

Conditions

Guidelines

Denaturation

Temp: 95ˇC. Time: 5 min on initial cycle; 30 seconds to 1 min on rest

Annealing

Temp: 5ˇC below Tm of primers; no lower than 40ˇC.  Time:  30-45 seconds.  This is the step where you would use a gradient.

Extension

Temp: 72ˇC.  Time:  ~1 min/kb of expected product; 5-10 min on last cycle.

Number of Cycles

~30 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 enter or proceed 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 (ˇC)

Time (min)

1

95

forever

2

95

5:00

3

95

0:30

4

45 – 65

0:30

5

72

1:00

6

Go to step 3, 34x

 

7

72

5:00

8

10

forever

 

The temperature gradient goes from left to right, left being the low end and right being the high end.  For example, in the above gradient, all of column one is 45ˇC, and all of column 12 is 65ˇC, with the columns in between being equally spaced between that. 

 

If, after you have tried both temperature and magnesium gradients (and checked all your reagents) your PCR still does not work, you can try using one of the following additives, listed in the order you should try them:

 

For specific instructions on how to enter your program into the thermocycler, see the manual for the thermocycler you want to use.  They are kept in the drawer to right of the machines.