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. 2019 Sep 5;9(17):e3347. doi: 10.21769/BioProtoc.3347

Table 3. PCR mix for 1st and 2nd PCR.

PCR step Reagent Concentration 1x 96x + 10%
1st PCR Nuclease -free water - 32.16 3376.80
PCR Buffer [10x] 4.00 420.00
Forward primer mix [50 μM each] 0.13 13.65
Reverse primer mix [50 μM each] 0.13 13.65
dNTPs [25 mM each] 0.40 42.00
HotStar Taq [5 U/μl] 0.18 18.90
subtotal mix 37.00 3885.00
cDNA template 3.00
Total 40.00
2nd PCR Nuclease-free water - 31.66 3324.30
PCR Buffer [10x] 4.00 420.00
Forward primer mix [50 μM each] 0.13 13.65
Reverse primer mix [50 μM each] 0.13 13.65
dNTPs [25 mM each] 0.40 42.00
HotStar Taq [5 U/μl] 0.18 18.90
subtotal mix 36.50 3832.50
DNA template 3.50
Total 40.00