Mouse Genotyping
Lab Protocols
Final PCR Reaction Conditions (5 µl volume)
- 25 ng genomic DNA
- 1X PCR Reaction Buffer (from enzyme manufacturer)
- 250 µM each dATP, dCTP, dTTP, dGTP
- 2.5 mM MgCl2
- 0.24 units of Taq Gold DNA Polymerase
(Applied Biosystems)
- 0.48 µM each primer
|
| RG25 |
|---|
| 95 °C |
12 min |
| 94 °C |
45 sec |
| 55 °C |
1 min. for 10 cycles |
| 72 °C |
1 min |
| 89 °C |
1 min |
| 55 °C |
1 min. for 15 cycles |
| 72 °C |
1 min |
| 72 °C |
10 min |
| 4 °C |
forever |
|
| RG5 |
|---|
| 95 °C |
12 min |
| 94 °C |
45 sec |
| 55 °C |
1 min for 10 cycles |
| 72 °C |
1 min |
| 89 °C |
1 min |
| 55 °C |
1 min for 25 cycles
|
| 72 °C |
1 min |
| 72 °C |
10 min |
| 4 °C |
forever |
|
| TD1 |
|---|
| 95 °C |
12 min |
| 94 °C |
20 sec |
| 62 °C |
20 sec for
20 cycles |
| 72 °C |
20 sec |
| 94 °C |
20 sec |
| 52 °C |
20 sec for 15 cycles;
touchdown to 52 °C
(~0.5 °C/cycle) |
| 72 °C |
10 min |
| 4 °C |
forever |
|
An average of 7 PCR products are pooled together using a Hydra 384 Microdispenser unit.
We used a capillary electrophoresis system for fragment separation.
