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Here's a tip we picked up from celebrity makeup artist Erik de Soto: Squeeze a small amount of primer onto your hand and blend it with a rosy liquid lipstick.
Furthermore, we reduced the amount of primer used in the spa-PCR to minimize the amount of unused primer after PCR.
Although secondary structure can hinder primer hybridization, the excess amount of primer
Our study showed that Exo I digestion was not efficient enough, leading to significant amount of primer dimers in the final product (data not shown).
By using a Taq polymerase with proofreading capability (AccuPrime Taq), we were able to significantly reduce the amount of primer dimer generated during the two-step PCR.
The percentage of primer extended in single nucleotide incorporations was determined by taking the ratio of extended primer to the total amount of primer (unextended primer + extended primer).
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Next, the 3'-end of this three-way junction acted as primer to trigger the strand displacement amplification (SDA), yielding a large amount of primers.
The total amount of primers increased with increasing assay number, but the primer concentration of each primer was maintained at 40 nM.
A common practice in mPCRs involving any non-amplification of a required gene ('weak locus') is to increase the amount of primers of the gene at same time with a decrease of the amount of primers for all the loci that can be amplified, especially those with strong amplifications.
Preferential amplification of one target sequence over another is a known phenomenon in mPCRs and it is usually overcome by increasing the amount of primers for the weaker amplification simultaneously with a decrease of primer concentrations for the stronger amplification.
For both types of ICLs, the amount of primers extended beyond the adducted site was less as compared to ND substrate; specifically, a 10- and 4-fold decrease was observed for N-dA-ICL1 and N-dA-ICL2, respectively.
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Justyna Jupowicz-Kozak
CEO of Professional Science Editing for Scientists @ prosciediting.com