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For 3′ RLM-RACE, all the reverse transcriptase steps were the same as 5′ RLM-RACE, except for the template where 1 μg of total RNA was used.
First, we conducted a RNA synthesis reaction with T7 RNA polymerase under the same NTP concentration and ratio between radioactive and nonradioactive GTP (1/1600) as the IAV reaction except for the template sequence.
The original concentration of the gel-extracted amplicons were restored by 10 PCR cycles with the same Ta and reaction mix as used for the previous PCR, except for the template, which was 2 µl gel-extract in this case.
To reduce variability among replicates, PCR premixes, which contain all reagents except for the template, were prepared and aliquotted into 0.2 ml thin well plates.
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Except for the templates in the 16 groups of Fig. 18, the majority has a very low number of representing reactions.
Single-stranded DNA (ssDNA) was generated by using the same conditions in multiplex PCR except for the templates that were 10 μl of the multiplex RT-PCR product.
Using four DNA duplexes with essentially the same sequence except for the templating bases, we measured the rate of incorporation of each correctly paired dNTP by WT Pol I KF) under single-turnover conditions.
Single-stranded DNA (ssDNA) was generated by using the same conditions in the multiplex PCR step except for the templates that were 2 μl of the multiplex RT-PCR product.
As for the origin of polymerases, I do not know of any ribozyme counterpart or RNA constituent for any known polymerase, except for the RNA template of telomerase.
Cut out the circle for the template.
Bscl2 sense and antisense cRNA probes were synthesized as previously described, except that the template for cRNA probe synthesis was amplified from m Bscl2 cDNA using primers m Bscl2e3F and m Bscl2e6R for PCR.
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