Table 2.
Details of Multiplex PCR for FLT3- ITD and TKD
| Primers- used for FLT3 -ITD & TKD gene |
Accession
number |
Produc
t size |
|
|---|---|---|---|
| 14F | 5’- TGT CGA GCA GTA CTC TAA ACA-3’ | NM_00411 9 |
366 bp |
| 15R | 5’- ATC CTA GTA CCT TCC CAA ACT C-3’ | NM_00411 9 |
|
| 20F | 5’ – CCG CCA GGA ACG TGC TTG- 3’ | NM_00411 9 |
114 bp |
| 20R | 5’ – GCA GAC GGG CAT TGC CCC- 3’ | NM_00411 9 |
|
| PCR reaction- was performed in a 25 µL reaction volume comprising of- | |||
| Reagent | Quantity | ||
| Taq Buffer | 2.5 µl | ||
| dNTP (0.2 mM) | 0.4 µl | ||
| Primers (10 pmol) |
0.3 µl of 14F and 15R and 0.4 µl of 20R and 20F | ||
| Taq polymerase | 0.5 µl | ||
| MgCl2 (1.5 mM ) | 0.1 µl | ||
| Autoclaved distilled water |
19.7 µl | ||
| cDNA | 1.5 µl | ||
| PCR conditions | |||
| Step | Temperature - time and number of cycles | ||
| Pre- Denaturation |
95°C -10 min –one cycle | ||
| Amplification -Denaturation -Annealing -Extension |
95°C - 30 sec 56°C - 45 sec x 40 cycles 72°C - 30 sec |
||
| Final Extension | 72⁰C - 10 minutes | ||
| Hold at 4⁰C | |||