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Table 2 Optimization of RNA hydrolysis while maintaining viral and plasmid DNA integrity

From: Alkaline hydrolysis to remove potentially infectious viral RNA contaminants from DNA

Sample ID Heat (min) DNA SCR RNA NaOH HCl TE SCR PCR ASF PCR CSF PCR DNA integrity
1- ASF DNA control 0 35 μl 7 μl 0 μl 0 μl 30 μl + + NA +
2- CSF DNA control 0 35 μl 7 μl 0 μl 0 μl 30 μl + NA + +
3- Plasmid DNA control 0 35 μl 7 μl 0 μl 0 μl 30 μl + NA NA +
4- ASF DNA heated 30 35 μl 7 μl 0 μl 0 μl 30 μl + + NA +
5- CSF DNA heated 30 35 μl 7 μl 0 μl 0 μl 30 μl + NA + +
6- Plasmid DNA heated 30 35 μl 7 μl 0 μl 0 μl 30 μl + NA NA +
7- ASF DNA treated 30 35 μl 7 μl 15 μl 15 μl 0 μl - + NA +
8- CSF DNA treated 30 35 μl 7 μl 15 μl 15 μl 0 μl + NA + +
9- Plasmid DNA treated 30 35 μl 7 μl 15 μl 15 μl 0 μl + NA NA +a
10- ASF DNA treated 45 35 μl 7 μl 15 μl 15 μl 0 μl - Incb NA +
11- CSF DNA treated 45 35 μl 7 μl 15 μl 15 μl 0 μl + NA + +
12- Plasmid DNA treated 45 35 μl 7 μl 15 μl 15 μl 0 μl - NA NA +a
13- ASF DNA treated 60 35 μl 7 μl 15 μl 15 μl 0 μl - Incb NA +
14- CSF DNA treated 60 35 μl 7 μl 15 μl 15 μl 0 μl - NA + +
15- Plasmid DNA treated 60 35 μl 7 μl 15 μl 15 μl 0 μl - NA NA +a
16- SCR in TE 0 0 μl 7 μl 0 μl 0 μl 65 μl + NA NA NA
17- SCR in salt 0 0 μl 7 μl 15μlc 15μlc 35 μl + NA NA NA
18- SCR undilute control 0 0 μl 2.5 μl 0 μl 0 μl 0 μl + NA NA NA
  1. Samples containing various combinations of ASF DNA, CSF cDNA, and plasmid DNA, under various treatment conditions. All samples contained SCR, and results for verification of SCR degradation and maintenance of plasmid DNA integrity are listed. NaOH and heat treatment incubation time used in this study range from 0 to 60 min. Following heat treatment for various times at alkaline pH, all samples were boiled for 10 min at neutral pH. aSlight nicking or denatured ssDNA was observed as compared to untreated control plasmid. bInconclusive rPCR result (Inc) due to high concentration of DNA template. cNeutralized salt solution (equal parts 1 N NaOH and 1 N HCl) reagent used as a positive control. NA indicates not analyzed