Qiagen Dneasy Blood and Tissue Kit Tutorial #1 скачать в хорошем качестве

Qiagen Dneasy Blood and Tissue Kit Tutorial #1

Notes Before Starting: *Perform all centrifugation steps at room temperature (15-25°C) *Redissolve any precipitates in Buffer AL and Buffer ATL *Add Ethanol to Buffer AW1 and AW2 concentrates before first use. *Equilibrate frozen tissue or cell pellets at room temperature. *Preheat an incubator to 56°C *Refer to the handbook for other materials such as fixed tissue, insects, bacteria, etc: https://www.qiagen.com/us/resources/d... *If you don't have any sterile H2O, here's how to make some:    • How To Sterilize H2O For Experiments? #Tut...   Protocol to follow: 1) Tissue preserved in 95% Ethanol: Cut 25mg (the size of half a grain of rice) of tissue such as liver, spleen or tail; wash them in sterile H2O to remove excess EtOH, and place them into 1.5 ml microcentrifuge tube. 2) Add 180 µl of Buffer ATL and 20 µl of Proteinase K. Mix by vortexing and incubate them at 56°C until completely lysed (it should take about 2-12 hours). Remove your tubes once every hour, vortex them, and put them back into the incubator. *If you are doing this process for the first time, it's better to incubate your tubes overnight (~8-12 hours). 3) Add 200 µl Buffer AL. *If you're using blood samples, after adding Buffer AL, incubate your tubes at 56°C for 10 minutes. 4) Add 200 µl Ethanol (96-100%). Vortex your tubes. 5) Transfer the mixture (This should be around 600µl of solution) into a DNeasy Mini spin column and place it in a 2 ml collection tube. Centrifuge at 8000 rpm for 1 minute. 6) Discard the flow through and collection tube. Place the spin column in a new 2 ml collection tube. 7) Add 500 µl Buffer AW1. Centrifuge at 8000 rpm for 1 minute. 8) Discard the flow through and collection tube. Place the spin column in a new 2 ml collection tube. 9) Add 500 µl Buffer AW2. Centrifuge at 14000 rpm for 3 minutes. 10) Discard the flow through and collection tube. Place the spin column in a new 1.5 ml collection tube. 11) Elute the DNA by adding 200 µl Buffer AE to the center of the spin column membrane. 12) Incubate at room temperature (15-25°C) for 1 minute. 13) Centrifuge for 1 minute at 8000 rpm. 14) Add 200µl Buffer AE again to increase DNA yield. 15) Incubate at room temperature (15-25°C) for 1 minute. 16) Centrifuge for 1 minute at 8000 rpm. 17) Transfer your DNA extract into a new 1.5 ml collection tube and label them properly. 18) Store them at -20C. For any questions, leave a comment.