Difference: ProtocolsEthanolPrecipitation (3 vs. 4)

Revision 42011-12-19 - JeffreyBarrick

 
META TOPICPARENT name="ProtocolList"
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Ethanol_precipitation

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Ethanol Precipitation

 
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Precipitating out salts from DNA samples

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>		Precipitating DNA/RNA from solution to remove salts and small nucleic acid fragments.
  Materials

  • 3M Sodium Acetate buffer, pH 5.2 (store at 4°C)
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  • Cold 100% Ethanol (-20°C)
  • Cold 70% Ethanol in sterile dH2O (-20°C)
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  • Cold 100% Ethanol (–20°C)
  • Cold 70% Ethanol in sterile dH2O (–20°C)
 
  • DNA sample
  • Linear acrylamide
  • 4°C Microcentrifuge (or normal microcentrifuge in cold room).

Procedure

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  1. Transfer DNA to a 500ul tube.
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  1. Transfer DNA to a 500µl tube.
 
  1. Add one tenth volume of Sodium Acetate buffer to equalize ion concentrations.
  2. Add at least two volumes of cold 100% ethanol.
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  1. Add 1-3ul linear acrylamide, mix, and let stand in -20°C freezer for at least one hour.***
  2. Centrifuge 14,000xg for 30 minutes at 4°C (to centrifuge we transferred reaction to a chilled 1.5ul tube).
  3. Remove as much supernatant as possible with a 1ml pipet; then remove the rest with a 200ul pipet.
  4. Add 200ul of cold 70% ethanol; centrifuge for 10 minutes at 4°C.
  5. Remove supernatant with a 200ul pipet; evaporate remaining ethanol in a 37°C water bath or heat block.
  6. Resuspend pellet in 50ul of water.
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  1. Add 1-3µl linear acrylamide, mix, and let stand in –20°C freezer for at least one hour.***
  2. Centrifuge 14,000×g for 30 minutes at 4°C (to centrifuge we transferred reaction to a chilled 1.5µl tube).
  3. Remove as much supernatant as possible with a 1ml pipet; then remove the rest with a 200µl pipet.
  4. Add 200µl of cold 70% ethanol; centrifuge for 10 minutes at 4°C.
  5. Remove supernatant with a 200µl pipet; evaporate residual ethanol using a speed-vac or by opening the tube and leaving at room temperature or in a 37°C heat block.
  6. Resuspend pellet in water or a new buffer of choice to an appropriate concentration.
 
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<		 * So for a 60ul ligation reaction: 60ul DNA, 6ul Sodium acetate, 120ul 100% EtOH, & 2ul acrylamide
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>		 * So for a 60µl ligation reaction: 60µl DNA, 6µl Sodium acetate, 120µl 100% EtOH, & 2µl acrylamide/
 
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<		(there's an interesting typo in the Ambion protocol for this... the acrylamide stock is 5mg/ml and the protocol says you want a final concentration of 10-20 g/ml linear acrylamide... I assume they meant ug/ml... 2ul of a 5mg/ml stock seems to be the concensus elsewhere online... that would be 10ug in 188ul or ~50ug/ml, but that was fine.)
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>		(there's an interesting typo in the Ambion protocol for this... the acrylamide stock is 5mg/ml and the protocol says you want a final concentration of 10-20 g/ml linear acrylamide... I assume they meant µg/ml... 2µl of a 5mg/ml stock seems to be the consensus elsewhere online... that would be 10µg in 188µl or ~50µg/ml, but that was fine.)
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-- LindseyWolf - 02 Dec 2011

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