Standard PCR Conditions
Promega Taq DNA Polymerase
| Component | Suggested Range | Starting point | For 20ul reation |
| 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 isn’t already in the buffer – if it is, don’t 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: 95°C. Time: 5 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 |
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 (°C) | 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 45°C, and all of column 12 is 65°C, 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:
- Bovine serum albumin (BSA): 10-100ug/ml
- DMSO: 1-10%
- Formamide: 1.25-10%
- PEG-6000: 5-15%
- Glycerol: 5-20%
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.
