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No polymerase or dNTPs were added to the mix.
It is important to emphasize that the picornaviral 3CDpro has no polymerase activity.
Currently, there is no polymerase chain reaction (PCR) assay available which differentiates between different species of Anaplasma or which can differentiate isolates of A. marginale within outbreaks and between different countries.
Limitations: No polymerase chain reaction (PCR) confirmatory studies have been completed.
Transcriptionally active states are those in which polymerase is bound at the promoter; inactive states have no polymerase bound.
Interestingly, in these conditions, no polymerase is found on miR-133b indicating that the PROX promoter does not direct transcriptional read-through into this region.
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In each case, the negative control with IgG non-immune antibodies, no polymerases or glycosylases immunoprecipitated, as expected.
Note that even when the gene is in the active state, it is possible that no polymerases are present (comparing black occupancy trace with red gene activity trace).
During gene transcription and translation (t = 1000 sec), the free polymerase probability density) is distributed among the five possible states from no polymerases bound (s001 = 301) to four polymerases bound (s001 = 297) with two polymerases bound (s001 = 299) being the most probable state.
Although no polymerases were involved, these SPR results are in line with the NMR and gel assay results as the greater S-conformation of the G*C T sequence posed a major hindrance toward the binding affinity.
Amplifications were performed in 10-μL volumes using 0.3 unit of GoTaq DNA polymerase (no. M300, Promega Corporation), 2 μL of 5× GoTaq PCR buffer (no. M7921, Promega Corporation), 10 50 ng of gDNA, 0.1 μL of 10 mmol/L dNTP mix (no. R0192, Thermo Fisher Scientific, Waltham, Massachusetts, USA), and 2 pmol of each primer.
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