Table 4.
Dilution of mono nuclear cells (MNCs) to obtain desired copy number of the beta-globin and RASSF1A genes that are homozygous (two copies per cell) in human MNCs.
| Sample (number of MNCs) | Gene copy number/μl (before DNA extraction) | Sample preparation for DNA extraction | Final elution volume in DNA Extraction (μl) | Gene copy number/μl (after DNA extraction) | Template volume for PCR (μl) | Final volume of PCR reaction (μl) | Final gene copy number/PCR reaction |
|---|---|---|---|---|---|---|---|
| Sample volume (μl) + DW (μl) | |||||||
| 1:10 Dilution (MNCs: 600) | 1200 | 83.3 + 16.7 | 50 | 2000 | 5 | 25 | 10,000 |
| 1:10 Dilution (MNCs: 600) | 1200 | 8.3 + 91.7 | 50 | 200 | 5 | 25 | 1000 |
| 1:100 Dilution (MNCs: 60) | 120 | 8.3 + 91.7 | 50 | 20 | 5 | 25 | 100 |
| To prepare the final gene copy number with 10 copies, we made a 1:10 dilution from the sample with the 20 copies (obtained after DNA extraction) | 5 | 25 | 10 | ||||