Application: DNA purification, PCR
Item #: 250-102-T
BioSite Brand: BioSite
Country Availability: all
Description: DirectPCR Lysis Reagent (mouse tail)
Provider: Nordic BioSite
Certain compounds in animal tissues inhibit PCR reactions. DirectPCR lysis reagents (patent pending) contain inhibitors of these PCR inhibitors. Therefore, DNA released into DirectPCR reagents is compatible with one-step PCR genotyping.
Product Type: Buffers and Mixes
- For a 0.5 cm tail, add 200-300 µl of DirectPCR Lysis Reagent (tail) containing 0.2-0.4 mg/ml of freshly prepared proteinase K. Proteinase K is stable in DirectPCR reagents for ~24 hours. If a small number of tails are processed and therefore the proteinase K powder is difficult to weigh, use genomic PCR grade proteinase K solution (Cat. No. 250-501-PK) at 0.5 -1.0 mg/mL (25-50 µL of proteinase K solution per 1 mL of DirectPCR reagent). See Table 1 for the initial conditions. NOTE: Although 200 µl of DirectPCR is usually sufficient for complete lysis of a 0.5 cm tail, applying 250-300 µl produces more reproducible results due to better mixing efficiency. Compare several different volumes of DirectPCR reagents for the best performance. If the tails do not mix well with the solutions, use 0.75 mL tubes
- Rotate tubes in a rotary hybridization oven at 55°C for 5 to 6 hours or until no clumps of tissue are observed. If necessary, rotation can be allowed overnight without loss of efficacy. Complete lysis is important. Since some tails may not be in contact with the solutions, replace the tails by shaking the bottles containing the tubes, preferably after 2-3 hours. NOTE: The rotary hybridization oven works better than the swing plate. Use 0.75 cm tubes for less than 200 µL of DirectPCR reagent. DNA fragmentation by prolonged spin will not significantly influence PCR performance. Use a roughly proportional volume of DirectPCR Lysis Reagent for samples of different sizes.
- Incubate crude lysates at 85°C for 45 min by floating the entire rack (containing the tubes) in a water bath. (Optional) Precipitate the hairs by centrifuging for 10 seconds prior to step 4. Crude lysates can be stored at -20 °C for 1 year (or at 4 °C for 1 week) without loss of efficacy.
- Use 0.5-1.0 μl of lysate for 50 μl of PCR reaction. DNA Rescue: DNA in crude lysates can be rescued for further analysis. Add NaCl to a final concentration of 250 mM and then add 0.7 volumes of isopropanol. DNA will form precipitates. Centrifuge at 4 °C for 2 min, discard the supernatant, wash the DNA with 1 mL of 70% EtOH, and dissolve the DNA in 50 µL of 10 mM Tris-HCl (8.0). Use 1 µl for PCR.
Research Area: Molecular Biology
Size: 500 Mouse tails (100 ml)
Species Reactivity: Mouse
1. Complete lysis. No large clumps of tissue should be seen after digestion. It is recommended to shake the flask (containing the microcentrifuge tubes) vigorously for 2-3 seconds at any time, once or twice, after the tissues begin to partially dissolve. This will physically disperse the partially digested tissues and relocate the microcentrifuge tube, in which the tails are separated from the lysis reagents, facilitating overall lysis efficiency.
2. Inactivation of proteinase K. Inactivation of proteinase K by incubating samples at 85-86°C for 45-50 min is essential to protect Taq polymerase from proteinase K.
3. Taq polymerase. We have tested many types of commercially available Taq polymerases. The enzymes listed are recommended for optimal results.
4. Fabric size. The size of the tails should be 0.5 cm or slightly smaller. Use a minimal volume (0.5-1 µl for 50 µl PCR reaction) of crude lysates for PCR amplification. Too many DirectPCR reagents inhibit PCR efficiency.
5. Small tubes and evaporation. To minimize evaporation, use a 0.75 mL tube when the reagent volume is less than 100 µL.
6. Small samples and dilution. If the volume of the DirectPCR reagent required is less than 50 µL, dilute the reagent up to 2-fold with water, maintaining the same proteinase K concentration. If the DirectPCR reagent is 2-fold diluted, apply more crude lysates for the PCR.
7. PCR machine. PCR machines are occasionally a source of technical problems.