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 point | For 20ul reation |
| Buffer | 1x | 4ul of 5x |
| Primers | 0.5 pMol | 0.5ul of each of 20 pMol |
| dNTP | 1mM (.25mM of each dNTP) | 0.5ul of 10mM |
|
Taq |
5U/ul units | 0.12ul |
| 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 mix | 1x | 10ul of 2x |
| Primers | 0.5 pMol | 0.5ul 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.
|
Conditions |
Guidelines |
|
Denaturation |
Temp: 95°C. Time: 2 min on initial cycle; 30 seconds to 1 min on rest |
|
Annealing |
Temp: 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. |
|
Extension |
Temp: 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.
|
Conditions |
Guidelines |
|
Denaturation |
Temp: 95°C. Time: 2 min on initial cycle; 30 seconds on rest |
|
Annealing |
Temp: 5°C below Tm of primers; no lower than 40°C. Time: 30 seconds. This is the step where you would use a gradient. |
|
Extension |
Temp: 72°C. Time: ~1 min/kb of expected product; 5 min on last cycle. |
|
Number of Cycles |
~35 cycles |
Cycle Conditions for Phusion Polymerase
|
Conditions |
Guidelines |
|
Denaturation |
Temp: 98°C. Time: 30 sec on initial cycle; 10 seconds on rest |
|
Annealing |
Temp: 5°C below Tm of primers; no lower than 40°C. Time: 20 seconds. This is the step where you would use a gradient. |
|
Extension |
Temp: 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 |
95 |
infinite |
|
2 |
95 |
2:00 |
|
3 |
95 |
0:30 |
|
4 |
45 – 65 |
0:30 |
|
5 |
72 |
1:00 |
|
Repeat 3-5 34x |
|
|
|
7 |
72 |
5:00 |
|
8 |
4 |
infinite |
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.