Standard PCR Conditions for Herman Lab
(not for Expand PCR)
| Component
| Suggested range
| We use
| for 50  l rxn:
__ ul of __ stock |
| primers
| 20-500 pM
| 50 pM
| 2.5 of 20pM/l
|
| buffer
| 1X
| 1X
| 5 of 10X
|
| dNTPs
| 20-500 M
| 200 M
| 5 of 2 mM
|
| MgCl2
| 1-9 M
| 2.5 M*
| 5 of 25 M
|
| Taq
| 1-2.5 units
| 2 units
| 0.5 l
|
| DNA template |
105-106
(units?) |
~50 ng genomic
~10 ng plasmid |
N/A
|
|
* unless it's in the buffer or you are varying the [Mg2+]
|
| Master Mix I |
Master Mix II |
| annealing |
5°C below lowest Tm of
primer or close to that, no lower than 40°C
|
| extension |
Temp: 72°C, Time: 1 min/Kb of
expected product, 10 min on last cycle |
| denaturation |
Temp: 95°C, Time: 5 min on initial
cycle and 30 sec to 1 min on rest |
| number of cycles |
25-30, usually 30 |
Notes:
- Primer stocks are 200 pmoles/l in TE pH 8.0
- Use sterile ddH2O for dilutions and reactions
- Filter tips should be used when getting into stocks (dNTPs, primers)
- Make 100 l dilutions of primers in 0.6 ml tube
|