Author: Gardner, Shea N.; Jaing, Crystal J.; Elsheikh, Maher M.; Peña, José; Hysom, David A.; Borucki, Monica K.
Title: Multiplex Degenerate Primer Design for Targeted Whole Genome Amplification of Many Viral Genomes Document date: 2014_8_3
ID: 1e44usl6_21
Snippet: Multiplexed primer sets were designed to cover the Nsp3 and 3 genes with 3 primer pairs per genomic region amplified when possible (total number of primers tested in two multiplex reactions was 53, Table S1 ). The primers were tested in the lab first by testing the primer pairs in individual reactions then as multiplexed reactions. No effort was made to optimize the PCR cycling conditions. RT-PCR conditions were as follows: reverse transcription .....
Document: Multiplexed primer sets were designed to cover the Nsp3 and 3 genes with 3 primer pairs per genomic region amplified when possible (total number of primers tested in two multiplex reactions was 53, Table S1 ). The primers were tested in the lab first by testing the primer pairs in individual reactions then as multiplexed reactions. No effort was made to optimize the PCR cycling conditions. RT-PCR conditions were as follows: reverse transcription was performed using random hexamers and the Superscript III RT reverse transcriptase kit (Invitrogen). The MHV-1 cDNA templates were amplified using the Q5 Hot Start High-Fidelity DNA Polymerase kit (New England BioLabs, Ipswich, MA), following manufacturer's instructions. PCR conditions consisted of 98 ∘ C for 30 s, followed by 35 cycles of 98 ∘ C for 10 s, 60 ∘ C for 20 s, and 72 ∘ C for 1 min. The final cycle was 72 ∘ C for 2 min.
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