DNA extraction protocol for Carageenophytes by rmpiloton

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									                            DNA extraction protocol for Carageenophytes
                                 Adapted from Wattier et. al 2000



      Incubate samples with 95 ul buffer and 5 ul enzyme (Promega Plant extraction Kit)
       overnight @ 50 OC 140 shakes/min.
      Grind samples with mortar and pestle.
      Put samples on ice while processing the rest of the samples.
      Add complete extraction buffer
      Incubate for 30 mins. @ 37 OC with 120 shakes/min.
      Spin at 13,000 rpm, 4 OC, for 15 mins.
      Slowly transfer supernatant to another eppy tube.
      Add 10 µl RNAse (2mg/ml)
      Incubate tubes for 30 mins. at 37 OC.
      Incubate tubes into ice for 30 mins. at 37 OC.
      Spin at 13,000 rpm, 4 OC, 15 mins.
      Transfer 750 ul of supernatant to another tube.
      Slowly add cold isopropanol (-30 OC).
      Incubate overnight at -30 OC.
      Spin at 13,000 rpm, 4 OC for 30 mins.
      slowly discard supernatant
      wash by adding 1 ml 70 % ethanol and spin at 3,000 rpm, 4 OC, for 10 mins. 3 times
      air-dry pellet and resuspend with 30 ul DNAse RNAse free distilled water.


Stock extraction Buffer:
100 mM Tris-HCl, 50 mM EDTA, 500 mM NaCl,
pH 8.0. Autoclave and store at room temperature.

Complete extraction Buffer:
For 50 samples
68 ml SEB
6.8 ml 20% SDS
125 µl Proteinase K stock sol’n (10 mg/ml)

								
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