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Potential biases of our experimental approach include more efficient amplification of certain bacteria versus others, and low sensitivity of the assay.
Studies have shown that addition of forward primers after the reverse transcription reaction and before activation of Taq DNA polymerase prevents the formation of primer-dimers and results in more efficient amplification [16].
They also made possible more efficient amplification of GC-rich templates than betaine and other previously described additives.
Otherwise, a more efficient amplification of methylated DNA would result in overestimated SHOX2 copy numbers in methylated specimens.
For other steps, TAQ DNA polymerase was used due to a more efficient amplification using this polymerase.
The latter might be due to the higher abundance of the transcript, or due to more efficient amplification of the transcript-3 primer set.
Similar(49)
We observed that smaller amplicons usually generate more RCs than larger ones, probably owing to more efficient PCR amplification for smaller DNA fragments (Supplementary Fig. S2B and S3A).
The high sequence similarity between of A. auriculiformis, A. mangium and their hybrids will allow more efficient cross amplification in gene isolation and characterization efforts.
Therefore, the careful optimization of reaction components and conditions will ultimately result in more reproducible and efficient amplification.
The amplification procedure described here can be optimized further, to allow for more efficient cell capture, higher amplification rates, and lower ADO rates.
We used the two-step Illumina adaptor design for the adapters and the PCR indexing primers because it decouples the indexing reaction from library amplification, and allows for more efficient bisulfite treated DNA library amplification and size selection.
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