Restriction enzyme digestion and gel electrophoresis

 

Our next goal is to setup restriction digests on the PCR products to quantify allelic polymorphisms. Recall that the recognition site for the HaeIII enzyme is GG|CC. In other words, the enzyme will only cut the DNA at these sequences.

 

Gel Casting (Gels will be used for both laboratory experiments)

 

  1. Each group of four (4) will prepare a 2% agarose gel.

 

  1. In a flask, combine 50 ml TBE buffer with 1 g agarose. Gently mix the solution.

 

  1. Microwave the buffer-agarose mixture at 30 sec intervals, mixing the solution between heating. Make sure you wear protective gloves as the flask will be hot! The agarose should be completely dissolved in the buffer and be clear.

 

  1. Add 10 ul SYBR Safe DNA stain to the hot flask and gently mix.

 

  1. Place two combs into each gel apparatus. Make sure the comb with 10 wells is placed facing down. Let the hot gel mixture cool a bit before pouring (~3 min). Carefully pour the gel into the gel tray, making sure that no liquid is spilled.

 

  1. Gels may be placed in the refrigerator to expedite solidification. This should take no more than 20 min.

 

 

DNA digestion

 

  1. Each student should obtain two new microcentrifuge tubes. Label one tube with your initials and the letter ‘U’ for undigested and one tube with the letter ‘D’ for digested.

 

  1. Transfer 5 ul of PCR product into tube labeled ‘U’ and 5 ul into tube labeled ‘D’.

 

  1. Add 1 ul of HaeIII enzyme to tube ‘D’.

 

  1. Briefly vortex or pipette-mix the mixture to homogenate.

 

  1. Incubate for 10 min at 37 °C.

 

  1. Save the remaining 15 ul of PCR product for DNA sequencing.

 

 

Gel electrophoresis

 

We will use gel electrophoresis to separate and visualize the DNA fragments resulting from digestion with the HaeIII restriction enzyme. Your goal is to compare the observed genotypes among individuals.

 

  1. When adequately solidified, place gel in gel apparatus. Make sure that the wells of the gel are near the negative (black) electrode.

 

  1. Each student should load two samples into the gel—one undigested sample and one digested with the restriction enzyme. Each gel should also include a DNA ladder to help size the fragments (load 5 ul of the DNA ladder). Below is a representative diagram of how the gels should be loaded for one group. U = undigested; D = digested.

 

  Marker   Student 1   Student 2       Student 3       Student 4  
            U D U D     U   D     U   D
                                                         
                                                         

 

 

  1. Run gel for 20 min at 120 V.

 

  1. Visualize results using transilluminator and obtain genotypes. Pay particular attention to your own banding pattern.

 

  1. Use your smartphone to take a picture of your gel.

 

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