DAY TWO- DNA digestion and gel preparation.

 

What do restriction enzymes do?

 

How does the DNA of a child compare to its parents? 

 

Put the steps in the proper order  to digest the DNA samples using the restriction enzyme, HindiIII.  You will use 5 uL of DNA for each sample.  Get your procedure approved by the teacher before you begin.  Remember to use fresh pipette tip for each transfer!

Step #

_____  Add 2 uL buffer to each tube.  Add 5 uL of the corresponding DNA to each tube.

_____  Put on goggles, gloves, and assemble all equipment

_____  Label 6 microfuge tubes M1+, M1-, B1+, B1-, S+, S- (+ will receive HindIII enzyme, - will not)

_____  Add 1 uL HindIII to all tubes receiving the enzyme (those labeled +)

_____  Add sterile H2O to bring total volume in tube to 20 uL

_____  Incubate at 37C for 30 minutes in the incubator block.

_____  Place all samples in the microcentrifuge and spin for five seconds to ensure all liquid is at bottom of tube.

 

Preparation of Gel-

            How would you prepare 50 mL of a 1% agarose solution?  (the solvent will be the 1XTAE buffer prepared previously.)  Show a correct mathematical setup, and write out the steps.

 

WORK:                                                           STEPS:

 

 

 

 

 

After the solution is prepared, heat it while stirring on the hot plate, until solution is no longer cloudy, and all agarose dissolves.  Set up the gel comb and gel box as directed, and pour agarose gel.  Allow to cool and harden.