Protocol for mtDNA Analysis and RFLP of Yellowtail (Sebastes flavidus),

Blue (Sebastes mystinus), and Olive (Sebastes serranoides) Rockfishes

 

Note:  Gloves must be worn at all times to reduce the risk of contaminating samples with human DNA.  Pipette tips must be discarded after each step.

 

DNA Extraction - Ruptures cells to release DNA

 

1)  Obtain a fin tissue sample that has been preserved in ethanol.

2)  Rinse with DI water, blot dry.

3)  Cut 20 mg of tissue into small pieces (about 1 mm).

4)  Place tissue in a labeled 1.5 ml microcentrifuge tube.

5)  Add 180 µl of Buffer ATL.

6)  Add 20 µl of Proteinase K. 

7)  Close the lid and vortex for 5 sec.

8)  Place sample tube in the heat block at 56oC overnight.

 

 

DNA Isolation -  Removes cellular debris

 

1)  Transfer the liquid portion to a clean 1.5 ml tube and discard any bone or sediment.

2)  Add 200 µl of Buffer AL to the sample.  Close the lid and vortex for 15 sec.

3)  Place the sample tube in the heat block at 70oC for 10 min.

4)  Remove the samples from the heat block and tap the tube on the counter to remove any droplets from the inside of the lid.

5)  Add 200 µl of ethanol (96-100%) to the sample. 

6)  Close the lid and vortex for 15 sec.  Tap the tube on the counter to remove drops inside lid.

7)  Transfer the sample to a QIAamp spin column in a 2 ml collection tube. Label the lid of the spin column.

8)  Close the lid and centrifuge for 1 min. at 8000 rpm.

9)  Place the spin column in a clean 2 ml collection tube.  Discard the used tube and liquid.

10)  Add 500 µl of Buffer AW1.

11)  Close the lid and centrifuge for 1 min. at 8000 rpm.

12)  Place the spin column in a clean 2 ml collection tube.  Discard the used tube and liquid.

13)     Add 500 µl of Buffer AW2.

14)     Close the lid and centrifuge for 3 min. at 14,000 rpm.

 

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15)  Place the spin column in a clean, labeled 1.5 ml microcentrifuge tube.  Discard the used tube and liquid.

16)  Add 200 µl of Buffer AE to the spin column.

17)  Let the sample stand at room temperature for 1 min.

18)  Close the spin column lid and centrifuge for 1 min. at 8000 rpm.

19)  Discard the spin column.

20)  Place the tube on ice for use in the PCR.

 

 

 

 

Polymerase Chain Reaction (PCR) - Makes copies of DNA sequence

(cytochrome b gene)

 

1)  Fill a cup with ice to hold the DNA sample and primers until ready to use.

2)  Label the PCR reaction tube, which contains the white buffer bead, on the side of the tube just under the lid. (Label location is important to prevent smearing of the ink during heating.)

3)  Add 18 µl of CB3RF/Gludg primer solution (10uM).

4)  Add 7 µl of isolated DNA.

5)  Close the lid and gently mix the contents of the tube.  Tap the tube on the table to return all liquid to the bottom.

6)  Place the tube in the PCR cycle machine and enter the following temperature program:

 

    4 temperatures, 1 hold, 36 cycles

    1 minute at 90oC

    1 minute at 94oC

    1 minute at 51oC

    1 minute at 72oC

    Hold at infinity at 4oC (set time to 99:59)

    Set volume to 25 ul

 

7)  Start program.

8)  Place the sample in the freezer when cycles are complete.

 

Restriction Enzyme - Cuts PCR product into fragments

 

1)  Label a 1.5 ml microcentrifuge tube.

2)  Add 9 µl of the Restriction Buffer solution.

3)  Add 10 µl of the PCR reaction (amplified mtDNA).

4)  Add 1 µl of StyI enzyme.

5)  Vortex for 15 sec. Tap gently on the counter to return the liquid to the bottom.

6)    Place in the heat block at 37oC for 1 hour.

 

 

Gel Electrophoresis - Shows lengths of DNA fragments

 

Gel Preparation:

1)  Weigh out 0.30 g of electrophoresis grade agarose and place it in an Erlenmeyer flask.

2)  Add 20 ml of TAE Buffer using a graduated cylinder.

3)  Microwave using medium power for 20 sec.  Swirl the flask to dissolve thoroughly.  If crystals are still present, microwave for 10 sec. more.

4)  Allow the agarose solution to cool until the flask bottom can be touched.

5)  Assemble the gel tank with the plastic boat, two gel dams, and a 15 tooth plastic comb.  (The comb must be placed on the negative (black terminal) end of the electrophoresis chamber.)

6)  Slowly pour the cooled gel into the chamber between the two dams.

7)  Allow the gel to solidify for 10-15 minutes.

8)  Once the gel is opaque, remove the two dams and the plastic comb.

 

 

Electrophoresis:

1)  Pour TAE Buffer in one end of the electrophoresis chamber until it reaches the top of the gel.  Repeat at the other end and pour until the gel is covered by buffer approximately 1 mm.

2)  Pipette 2 µl of the 100 bp ladder into the first and last wells on the gel.

3)  Add 4 µl of loading dye to the tube containing the restriction product.

4)  Pipette 8 µl of the restriction product into one of the wells, note its location.

5)  Place the cover on the chamber and connect the wires to the power supply.  (Ensure that the red and black leads are correctly connected on both the chamber and the power supply.)

6)     Follow the directions on the power supply to start the electrophoresis.

7)     Run the gel for about 45 minutes at 85 V.

 

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Staining (Wear gloves and goggles):

1)  Turn off the power supply and disconnect the power cord from the chamber.

2)  Carefully remove the gel from the plastic boat and place it in ethidium bromide for 10 min.

3)  Using a spatula, move the gel to the water rinse bath for 5 min.

4)  Remove the gel from the rinse bath and place it right-side-up in the Gel-Doc.

5)  Follow the instructions on the Gel-Doc to view the DNA banding pattern and obtain a print of the gel.

6)  Using the banding pattern, identify the species of rockfish (see Page 6).

 

 

PCR Cleanup ­ purifies PCR product for sequencing

 

1)  Label a 1.5 ml microcentrifuge tube.

2)  Add 50 µl of Buffer PB.

3)  Add 10 µl of PCR product and vortex for 15 sec.

4) Transfer the sample to a QIAamp spin column in a 2 ml collection tube. Label the lid of the spin column.

5)  Close the lid and centrifuge for 1 min. at 8000 rpm.

6)  Place the spin column in a clean 2 ml collection tube.  Discard the used tube and liquid.

7)  Add 750 µl of Buffer PE.

8)  Close the lid and centrifuge for 1 min. at 8000 rpm.

9)  Discard the liquid and centrifuge for an additional 1 min. at 14,000 rpm

10)  Place the spin column in a clean, labeled 1.5 ml microcentrifuge tube.

11)  Add 30 µl of Buffer EB.

12)  Close the spin column lid and let the sample stand at room temp. for 1 min.

13)  Centrifuge for 1 min. at 14,000 rpm.

14)  Discard the spin column. (Keep the liquid sample)

 

 

Cycle Sequence Reaction ­ amplifies DNA for sequencing using a single primer

 

1)  Label 2 PCR tubes on the side, just below the lid.  (Label location is important to prevent smearing of the ink during heating.)

2)  Add 2 µl of Big Dye Terminator to each tube.

3)  Add 4 µl of clean PCR product to each tube.

4)  Add 3 µl of CB3RF primer to one of the tubes (1 µM).

5)  Add 3 µl of Gludg primer to the remaining tube (1µM).

6)  Close the lid and gently mix the contents of the tube.  Tap the tube on the table to return all liquid to the bottom.

7)  Place the tubes in the thermocycler and enter the following temperature program:

 

    3 temperatures, 1 hold, 25 cycles

    10 seconds at 96oC

    5 seconds at 50oC

    4 minutes at 60oC

    Hold at 4oC for infinity  (set time to 99:59)

    Set volume to 12 µl

 

8)  Start program.

9)  Place the sample in the freezer when cycles are complete.

 

 

 

 

File written by Adobe Photoshop® 4.0

 

Banding patterns for rockfish RFLP can be used to identify species.