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Based on the sequences of differential products isolated from RDA experiments, 10 pairs of PCR primers were designed for 10 groups of candidate gene sequences.
As shown in Fig. 1, RDA differential products were isolated and DNA was extracted from each of the five bands obtained from the loss side of the third round of hybridization, and DNA from each band was cloned separately.
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After subtractive hybridization, the male-specific DNA was re-amplified to generate the Differential Product 1 (DP1) using J adaptors as primers.
The male specific bands present in the Differential Product 3 (DP3) were gel eluted and cloned into the PCR®2.1-TOPO® vector using the TOPO TA cloning kit (Invitrogen).
Following this procedure, a second and a third round of subtractive hybridizations were performed: the Differential Product 2 (DP2) was obtained using a new DNA tester generated by substituting the DP1 J adaptors for N adaptors and a 1/800 tester/driver ratio, while the Differential Product 3 (DP3) was obtained substituting the DP2 N adaptors for J adaptors and a 1/40000 tester/driver ratio.
One hundred clones from each DNA band were saved and a subset of 100 clones (20 clones from each differential product) was selected randomly for further characterization.
As a result decertification decisions appear to be significantly affected by a larger number of revenue, cost, and nonfinancial factors (that is, output price differentials, product and market diversification, input prices, and institutional factors).
Conclusively, estimating price dependence while considering pork as a non-differentiated product, would ignore differential responses.
There was no consistent price differential for products made with different resins.
Differential cDNA products were used as probes to screen zebrafish cDNA libraries.
To identify the differential amplification products, corresponding bands were excised, reamplified, cloned, and subject to DNA sequencing.
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