DNA Preparation from Tail Clip: Difference between revisions

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Use Qiagen DNEasy kit for Blood and Tissue Samples
Use Qiagen DNEasy kit for Blood and Tissue Samples.  For a crude preparation, often good enough for PCR genotyping see [[Preparation of Tail Samples (for Genotyping)]]


[edit] Protocol
==Protocol==


1. Cut .5cm lengths of tail into a 1.5 ml microcentrifuge tube. Add 180ul Buffer ATL.
#Cut .5cm lengths of tail into a 1.5 ml microcentrifuge tube. Add 180ul Buffer ATL.
2. Add 20ul proteinase K. Mix by vortexing and incubate at 56C until tissue is completely lysed. (Overnight)  
#Add 20ul proteinase K. Mix by vortexing and incubate at 56C until tissue is completely lysed. (Overnight)  
3. Vortex for 15s. Add 200ul Buffer Al to sample and vortex. Then add 200 ul ethanol (96-100%) and vortex.
#Vortex for 15s. Add 200ul Buffer Al to sample and vortex. Then add 200 ul ethanol (96-100%) and vortex.
4.Pipet the mixture from step 3 into DNeasy Mini spin column. Centrifuge at 8000rpm for 1 minute. Discard flow-through and collection tube.  
#Pipet the mixture from step 3 into DNeasy Mini spin column. Centrifuge at 8000rpm for 1 minute. Discard flow-through and collection tube.  
5. Place DNeasy Mini spin column in new collection tube and add 500 ul Buffer AW1. Centrifuge for 1 minute at 8000rpm. Discard flow-through and collection tube.  
#Place DNeasy Mini spin column in new collection tube and add 500 ul Buffer AW1. Centrifuge for 1 minute at 8000rpm. Discard flow-through and collection tube.  
6. Place DNeasy Mini spin column in new collection tube and add 500 ul Buffer AW2. Centrifuge for 3 minutes at 14000rpm. Discard flow-through and collection tube.  
#Place DNeasy Mini spin column in new collection tube and add 500 ul Buffer AW2. Centrifuge for 3 minutes at 14000rpm. Discard flow-through and collection tube.  
7. Place DNeasy Mini spin column in clean 1.5 microcentrifuge tube and pipet 200ul Buffer AE directly onto the DNeasy membrance. Incubate at room tempature for 1 minute and then centrifuge for 1 minute at 8000rpm to elute.
#Place DNeasy Mini spin column in clean 1.5 microcentrifuge tube and pipet 200ul Buffer AE directly onto the DNeasy membrance. Incubate at room tempature for 1 minute and then centrifuge for 1 minute at 8000rpm to elute.
 
[[Category: Mouse Work]]
[[Category: Molecular Biology]]
[[Category: DNA]]
[[Category: Genotyping]]

Latest revision as of 19:23, 24 January 2026

Use Qiagen DNEasy kit for Blood and Tissue Samples. For a crude preparation, often good enough for PCR genotyping see Preparation of Tail Samples (for Genotyping)

Protocol

  1. Cut .5cm lengths of tail into a 1.5 ml microcentrifuge tube. Add 180ul Buffer ATL.
  2. Add 20ul proteinase K. Mix by vortexing and incubate at 56C until tissue is completely lysed. (Overnight)
  3. Vortex for 15s. Add 200ul Buffer Al to sample and vortex. Then add 200 ul ethanol (96-100%) and vortex.
  4. Pipet the mixture from step 3 into DNeasy Mini spin column. Centrifuge at 8000rpm for 1 minute. Discard flow-through and collection tube.
  5. Place DNeasy Mini spin column in new collection tube and add 500 ul Buffer AW1. Centrifuge for 1 minute at 8000rpm. Discard flow-through and collection tube.
  6. Place DNeasy Mini spin column in new collection tube and add 500 ul Buffer AW2. Centrifuge for 3 minutes at 14000rpm. Discard flow-through and collection tube.
  7. Place DNeasy Mini spin column in clean 1.5 microcentrifuge tube and pipet 200ul Buffer AE directly onto the DNeasy membrance. Incubate at room tempature for 1 minute and then centrifuge for 1 minute at 8000rpm to elute.
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