Buffers & Mixes

DirectPCR Lysis Reagent (cell)

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DirectPCR Lysis Reagent (cell)

Article No

250-301-C

Application

DNA Purification, PCR

Size

Cultured cell (50 ml)

Product insert

Specifications

Application DNA Purification, PCR
Article No 250-301-C
BioSite Brand BioSite
Country Availability all
Description DirectPCR Lysis Reagent (cell)
Supplier Nordic BioSite
Notes Certain compounds in animal tissues and cells 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. Suspend cells from a 10 cm plate in 200–300 μl DirectPCR Lysis Reagent (Cell) containing freshly prepared 0.2-0.4 mg/ml Proteinase K. Proteinase K is stable in DirectPCR reagents for ~24 hrs. If a small number of cells 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). NOTE: For a small number of cells, dilute DirectPCR Reagent with distilled water up to 10 times and then use a proportionally larger volume of lysates for PCR (see Table 1). 2. Rotate the tubes in rotating hybridization oven at 55°C for 5-6 hrs or until no clumps are observed. If necessary, rotation can be allowed overnight without loss of efficacy. Complete lysis is important. For more reproducible results, re-position once the tubes by shaking the bottles containing tubes, preferentially after 2-3 hrs. NOTE: Rotating hybridization oven performs better than rocking plate. Use 0.75 cm tubes for less than 150 μl of DirectPCR Reagent (Cell). 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 Cultured cell (50 ml)
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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