| Reagent / Constituent | Concentration | Volume (µL) |
|---|---|---|
| buffer (Qiagen) | 10X | 2.0 |
| MgCl2 (Qiagen) | 15 mM | 0.8 |
| dNTPs (Qiagen) | 2.5mM | 1.6 |
| Primer 1 Name: EGFP forward primer | 50 µM | 0.2 |
| Primer 2 Name: EGFP reverse primer | 50 µM | 0.2 |
| Primer 3 Name: Actin PCR control/forward | 50 µM | 0.4 |
| Primer 4 Name: Actin PCR control/reverse | 50 µM | 0.4 |
| Tag Polymerase (Qiagen) | 0.2 | |
| Q solution (Qiagen) | 4.0 | |
| Water | 9.2 | |
| tail genomic DNA sample | 1.0 | |
| Total Volume of Reaction: | 20.0 µL | |
| # | Primer Name | Nucleotide Sequence (5' - 3') |
|---|---|---|
| 1 | EGFP forward primer | CCT ACG GCG TGC AGT GCT TCA GC |
| 2 | EGFP reverse primer | CGG CGA GCT GCA CGC TGC GTC CTC |
| 3 | Actin PCR control/forward primer | GAT GAC GAT ATC GCT GCG CTG GTC G |
| 4 | Actin PCR control/reverse primer | GCC TGT GGT ACG ACC AGA GGC ATA CAG |
| Step | Temp. (°C) | Delta per cycle | Time(secs.) | # of Cycles |
|---|---|---|---|---|
| 1. Initial melting | 94 | - | 180 | 1 |
| 2. Denaturization | 94 | - | 30 | - |
| 3. Anealing | 60 | - | 45 | - |
| 4. Elongation | 72 | - | 45 | - |
| Repeat steps 2, 3, 4 | total cycles: 30 | |||
| 5. Amplification | 72 | - | 600 | 1 |
| Finish | 4 | - | indefinite | n/a |
Electrophoresis:
2.5% Agarose gel at 200-250 mVolts for 30-45 mins.
| Band # | Position | Genotype |
|---|---|---|
| 1 | ~300 bp | EGFP |
| 2 | 1 kb | Actin (control) |
Last Modified: April 04, 2005
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Last Modified: April 20, 2005