100pmol/ul is a 100uM solution.(pmol/ul = nmol/ml = umol/L……uM)
If you use 2.5ul of the primer solution into a final volume of 25ul the calculation is as follows.
V1 x C1=V2 x C2 where V= volume in uL, C=concentration in uM
2.5uL x 100uM = 25uL x C2
(2.5 x 100)/25=C2
10uM final primer concenration
(Note: it is important to keep units the same on both sides of =)
It is confusing that 2 of 5 did work, but you might check the MgCl2 concentration in the new Mastermix it may be at a different concentration. Did the 3 samples that failed give any product?? How much DNA do you add?
Hope this helps,
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