Difference between revisions of "DNA Preparation from Tail Clip"
From Bridges Lab Protocols
Davebridges (Talk | contribs) m |
|||
| (2 intermediate revisions by one other user not shown) | |||
| Line 1: | Line 1: | ||
Use Qiagen DNEasy kit for Blood and Tissue Samples | Use Qiagen DNEasy kit for Blood and Tissue Samples | ||
| − | + | ==Protocol== | |
| − | + | #Cut .5cm lengths of tail into a 1.5 ml microcentrifuge tube. Add 180ul Buffer ATL. | |
| − | + | #Add 20ul proteinase K. Mix by vortexing and incubate at 56C until tissue is completely lysed. (Overnight) | |
| − | + | #Vortex for 15s. Add 200ul Buffer Al to sample and vortex. Then add 200 ul ethanol (96-100%) and vortex. | |
| − | + | #Pipet the mixture from step 3 into DNeasy Mini spin column. Centrifuge at 8000rpm for 1 minute. 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. | |
| − | + | #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 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. | |
Latest revision as of 17:58, 5 June 2009
Use Qiagen DNEasy kit for Blood and Tissue Samples
Protocol
- Cut .5cm lengths of tail into a 1.5 ml microcentrifuge tube. Add 180ul Buffer ATL.
- Add 20ul proteinase K. Mix by vortexing and incubate at 56C until tissue is completely lysed. (Overnight)
- Vortex for 15s. Add 200ul Buffer Al to sample and vortex. Then add 200 ul ethanol (96-100%) and vortex.
- Pipet the mixture from step 3 into DNeasy Mini spin column. Centrifuge at 8000rpm for 1 minute. 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.
- 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 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.
