Case Of The Crown Jewels Template Page 10
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28Overview of Laboratory Protocol
Each student receives three DNA samples, one from the crime scene and one from each of the two
suspects. Tell students that they will now apply the concepts they have learned in the pre-laboratory
activity to the real DNA in the laboratory. The crime samples need to be prepared by the instructor
before beginning the laboratory activity; instructions for the crime samples follow the laboratory
overview protocol.
Step 1 – Mixing the DNA with restriction enzyme
Add the DNA, water, buffer and enzyme together in a microcentrifuge tube and spin in a
microcentrifuge for five seconds to collect the contents at the bottom of the tube. Incubate the
samples at 37°C.
NOTE: This may be a stopping point. Samples may be removed from the incubator and stored
at 2 to 8°C (refrigerated) for up to 24 hours. Samples may be stored at –15 to -25°C (frozen) for
up to one week.
Step 2 – Prepare a 1 % agarose gel
If available, use EGels to run the samples. EGels are convenient pre-prepared agarose gels that
come with blue stain or ethidium bromide. Follow the manufacturer’s recommendations for
loading and running the gels.
If EGels are not available, prepare a one percent agarose gel by combining 1 g of agarose for
every 100 mL of 1X TAE. Heat in a microwave until the agarose has completely dissolved. Add
ethidium bromide if that is the stain the class will use (10 mg/mL) to a final concentration of 0.2
µg/mL and mix. If methylene blue will be used to stain the gel (see step 5), do not add
ethidium bromide to the agarose.
Allow the agarose to cool to approximately 55 °C. Prepare and seal the ends of the gel mold
according to the manufacturer’s instructions. Also position the desired comb to cast the wells.
Pour the cooled, liquefied agarose into the gel mold and allow it to solidify. Gels should be 5 –0
8 mm thick.
After the gel has solidified place it in the electrophoresis chamber and carefully remove the
comb. Add a sufficient volume of the 1X TAE buffer used to make the gel to the electrophoresis
chamber to cover the gel by 1 – 2 mm.
Step 3 – Prepare and load the DNA samples
Add loading dye to the DNA samples and place in a 65°C heating block for five minutes. Put 15
µL of the crime DNA and 15 µL of each suspect DNA into separate wells on the gel.
Loading dye is not necessary if the samples are run on the blue stain EGel.
Case of the Crown Jewels
Page 8
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