Table 1.

Details of PCR for NPM1

Primers- were used for exon 12 of NPM1 gene Bold letters depict mismatch introduced (C to G and C to A mutation)		Position Accession number NM_002520	Product size
NM-F2	5’ – ATC AAT TAT GTG AAG AAT TGC TTA C-3’	901–925	349 bp
NPM-Rev6	5’ – ACC ATT TCC ATG TCT GAG CAC C- 3’	1249–1228
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)	forward = 0.2 µl reverse = 0.2 µl
Taq polymerase	0.4 µl
MgCl2 (2 mM )	0.1 µl
Autoclaved distilled water	19.2 µl
cDNA	2 µl
PCR conditions
Step	Temperature - time and number of cycles
Pre-Denaturation	95°C - 5 min x one cycle
Amplification -Denaturation -Annealing -Extension	95°C - 30 sec 56°C - 45 sec x 35 cycles 72°C - 30 sec
Final Extension	72⁰C - 7 minutes
Hold at 4⁰C