DNA Isolation Lab.

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Presentation transcript:

DNA Isolation Lab

COLLECT AND LYSE CELLS Fill 15 mL test tube with 3 mL of water Label with your initials. GENTLY chew on the inside of your cheek to loosen cells. Images from BIO-RAD lab manual

COLLECT AND LYSE CELLS Use the 3 mL of water in tube to rinse your mouth and SPIT liquid into 15 mL tube or cup. DON’T SWALLOW LIQUID! http://www.scienceprogress.org/2008/10/spitomics/

COLLECT AND LYSE CELLS Using a new clean pipette, ADD 2 mL of LYSIS BUFFER to your 15 mL tube. Place cap on tube. GENTLY INVERT 5 TIMES to lyse cells. DON’T SHAKE THE TUBE! Images from BIO-RAD lab manual

REMOVE PROTEINS Add 5 DROPS of PROTEASE + SALT solution REMOVE PROTEINS Add 5 DROPS of PROTEASE + SALT solution. Place cap on tube. GENTLY INVERT 5 TIMES to mix DON’T SHAKE THE TUBE! Images from BIO-RAD lab manual

http://www. directindustry http://www.directindustry.com/prod/stuart-equipment/laboratory-test-tube-block-heaters-63431-412378.html REMOVE PROTEINS Place tube in 50° C WATER BATH for 10 minutes to allow protease to work. Images from BIO-RAD lab manual

MAKING THE DNA VISIBLE Fill a disposable pipette with ICE COLD ALCOHOL MAKING THE DNA VISIBLE Fill a disposable pipette with ICE COLD ALCOHOL. Hold tube at a 45 degree angle. Add alcohol by SLOWLY dispensing it down the inside wall of the tube so it forms a layer on top. It will take repeated additions to add 10 mL. Images from BIO-RAD lab manual

Screw cap on tube and place upright in test tube rack Screw cap on tube and place upright in test tube rack. Leave it undisturbed at room temperature for 5 minutes. After 5 minutes look at tube (especially in area where alcohol and cell extract layers meet) http://www.arssales.com/equine/assets/images/test_tube_rack.jpg

TIGHTLY CAP TUBE and SLOWLY INVERT 5 times. http://learn.genetics.utah.edu/content/labs/extraction/howto/ After 5 minutes look at tube (especially in area where alcohol and cell extract layers meet) TIGHTLY CAP TUBE and SLOWLY INVERT 5 times. . The stringy white material is DNA. The salt you added helps it stick together DNA stays dissolved in water, but when salty DNA comes in contact with alcohol It “undissolves”. Images from BIO-RAD lab manual

Dry off the opening of the tube and seal with “Crazy glue” Images from BIO-RAD lab manual Use a disposable pipette to transfer your DNA into a vial along with 750 uL – 1 mL of alcohol. Dry off the opening of the tube and seal with “Crazy glue” http://paint-and-supplies.hardwarestore.com/50-272-super-glue.aspx