Manual Extraction of Cell Lines


 * 1) The cells you recieve will probably be suspended in ethanol. Pipette about 800ul of the cell in ethanol liquid into a microcentrifuge tube. Make sure the centrifuge is balanced, and centrifuge at 300 x g for about 5 min.  You should be able to see a white solid at the bottom.
 * 2) Pipette 200ul of PBS (phosphate buffer saline which may be found in the fridge in the lab) into the microcentrifuge tube with your cells to resuspend them.
 * 3) Add 20ul proteinase K.
 * 4) Add 200ul Buffer AL (without ethanol added) and vortex it until it looks mixed and put it in the incubator at 56°C for 10 minutes.
 * 5) Add 200ul ethanol (96-100%) and vortex until it is homogeneous.
 * 6) Pipette the mixture (set the pipette to 600ul) into a spin column and make sure to label the cap, and centrifuge it at 8000 rpm for 1 min (you have to set the centrifuge to 2 min, and once it reaches 8000 rpm start your watch and time it for 1 min).
 * 7) Throw out the collection tube full of liquid and put the spin column into a new tube. Add Buffer 500ul AW1 and centrifuge at 8000 rpm for 1 min.
 * 8) Throw out the collection tube full of liquid and put the spin column into a new tube. Add Buffer 500ul AW2 and centrifuge at 130 000 rpm for 5 min.
 * 9) Throw out the collection tube full of liquid and remove the spin column carefully as not to let it touch the liquid and put the spin column into a 1.5 or 1.7 ml microcentrifuge tube (not from the kit, one with a cap) and label it.
 * 10) Add 100ul Buffer AE (elution buffer) and let it sit at room temperature for 1 min. Centrifuge at 8000 rpm for 1 min.
 * 11) The liquid in the bottom of the microcentrifuge tube is your DNA.

Return to Manual Extraction.

The next step is the PCR Procedure.

Updated May 3 2009