Buffers & Mixes

DirectPCR Lysis Reagent (mouse ear)

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DirectPCR Lysis Reagent (mouse ear)

Article No

250-402-E

Application

DNA Purification, PCR

Species Reactivity

mouse

Size

500 mouse ears (50 ml)

Product insert

Specifications

Application DNA Purification, PCR
Article No 250-402-E
BioSite Brand BioSite
Country Availability all
Description DirectPCR Lysis Reagent (mouse ear)
Supplier Nordic BioSite
Notes Certain compounds in animal tissues inhibit PCR reactions. DirectPCR Lysis Reagents (Patent Pending) contain inhibitors of these PCR inhibitors. Therefore, DNA released in DirectPCR reagents is compatible for one-step PCR genotyping.
Product Type Buffers & Mixes
Protocol 1. For 0.2 cm diameter-ear piece, add 100–200 μl DirectPCR Lysis Reagent (Ear) containing freshly prepared 0.2-0.4 mg/ml Proteinase K. It is important to use 0.5-0.75 ml tube for ears. Proteinase K is stable in DirectPCR reagents for ~24 hrs. If a small number of ears are processed, and therefore it is difficult to weigh Proteinase K powder, use genomic PCR-quality Proteinase K solution (cat # 250-501-PK) at 0.5-1.0 mg/ml (25-50 μl Proteinase K solution per 1 ml DirectPCR reagent). See Table 1 for starting conditions. NOTE: Although 100 μl DirectPCR is usually sufficient for complete lysis of 0.2 cm ear piece, application of 150-200 μl yields more reproducible results because of better mixing efficiency. Compare several different volumes of DirectPCR reagents for best performance. 2. Rotate the tubes in rotating hybridization oven at 55°C for 5-6 hrs or until no tissue clumps are observed. If necessary, rotation can be allowed overnight without loss of efficacy. Complete lysis is important. Since some ear pieces may not be in contact with solutions, re-position once the sample by shaking the bottles containing tubes, preferentially after 2-3 hrs. NOTE: Rotating hybridization oven performs better than rocking plate. DNA fragmentation by prolonged rotation will not influence significantly PCR performance. Use roughly proportional volume of DirectPCR Lysis Reagent for different sized samples. 3. Incubate crude lysates at 85°C for 45 min by floating the whole rack (containing tubes) on a water bath. (Optional) Precipitate hairs by centrifuging for 10 sec before step 4. Crude lysates may be stored at -20°C for 1 year (or at 4°C for 1 week) without losing efficacy. 4. Use 0.5-1.0 μl of lysate for 50 μl PCR reaction. Rescue of DNA: DNA in crude lysates can be rescued for further analysis. Add NaCl to a final concentration of 250 mM, and then add 0.7 volume of isopropanol. DNA will form precipitates. Centrifuge at 4°C for 2 min, discard supernatant, wash DNA with 1 ml 70% EtOH, and dissolve DNA in 50 μl 10 mM Tris-HCl (8.0). Use 1 μl for PCR.
Research Area Molecular Biology
Size 500 mouse ears (50 ml)
Species Reactivity mouse
Storage RT
Technical Specifications Important Technical Tips 1. Complete lysis. Big tissue clumps should not be observed after digestion. It is recommended to vigorously shake the bottle (containing microfuge tubes) for 2-3 sec anytime, once or twice, after tissues begin to partially dissolve. This will physically disperse partially digested tissues and reposition microfuge tube, in which tails are separated from lysis reagents, thereby facilitating overall lysis efficiency, 2. Proteinase K inactivation. Inactivation of proteinase K by incubating samples at 85C-86C for 45-50 min is critical to protect Taq polymerase from proteinase K. 3. Taq polymerase. We have tested many types of commercially available Taq polymerases. The listed enzymes are recommended for optimal results. 4. Tissue size. The size of 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 much 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 required DirectPCR reagent volume is less than 50 μl, dilute the reagent by up to 2-fold with water, while maintaining the same concentration of proteinase K. If the DirectPCR reagent is '2-fold' diluted, apply '2-fold' more crude lysates for PCR reaction. 7. PCR machine. PCR machines are occasionally a source of technical problems.
Product Page Updated 2018-07-18T09:23:28.417Z

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