i have synthesized RACE ready cDNA using SMART RACE Kit from Clontech. Universal Primer Mixture A (UPM) provided with kit was finished. So we order to synthesize new primers. we dissolve both of the primers and make a mixture as advised in Kit. but the these primers do not work with the same cDNA and always give smears. i try to standardize at different temp and MgCl2 conc.
Long UPM stock = 100uM
Short UPM stock = 100uM
4ul of Long UPM + 20ul of Short UPM + 76 ul water= 100ul
use 2.5 ul of this in 25 ul reaction mix to get final 0.4 uM of Long and 2uM of short UPM
Have i made any mistake to prepare working primer mix?
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