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The biochemical mechanism that determines product length is not fully characterized.
The final product length is 155 bp.
The PCR product length was approximately 400 bp (figure 5a) and was subsequently sequenced (figure 5b).
The sequence of oligonucleotides and the product length are shown in table 1.
Primer pairs (TGAGGTCTTCATCTCCTT/AAACCAACCTCTCCGTGT for PDR5-1, the PCR product length is 1111 bp, GCAAGACCTTCCTCTCCT/CAATTTCTCCGTAAGT ATCG for PDR5-2, the PCR product length is 1114 bp) were used to detect transcription of TMDR1 and TMDR2 constructs.
We tested with PCR for desired product length and specificity; no pseudogenes were amplified (Figure 2 shows the PCR results).
The primers were tested using 1ul of the sheared single cell lysates that was MDA-amplified to verify the correct product length and product specificity.
For example, the melting temperature, the primer pair length, the GC content, the PCR product length, the secondary structure, and the specificity were all considered.
The following oligonucleotide primers were designed with PRIMER3 software [32] and were selected such that PCR product length did not exceed 210 bp.
The constraints used are the followings: 1) melting temperature, 2) primer length and GC content, 3) PCR product length, 4) secondary structure, 5) specificity, and 6) terminal limitation.
We also performed RT-PCR using each sample and a primer specific for myosin heavy chain (MHC) IIb (product length, 197 bp).
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