Protocols for Experiment 2

PROTOCOLS FOR EXPERIMENT 2 (Labs 4, 5 and 6)

Lab 4: Extraction of DNA from mouse tissues, agarose gel electrophoresis and amplification of polymorphic markers.

DNA extraction of mouse DNA:

You will be given 7 samples to extract DNA from.

Sample 1: General Hans Canis
Sample 2: Field Marshall Heinrich Rattus
Sample 3: Mrs. Heidi Rattus
Sample 4: Miss. Gertrude Musca
Sample 5: Mr. Ivan Apis
Sample 6: Blood on the envelope
Sample 7: Hair found in Gertude Musca's apartment.

Label your tubes carefully and make sure you don't mix up your samples as that will lead to deduction in marks.

Protocol for DNA extraction from mouse tissues

1.) Label your samples.

2.) Add 200 ul of DNAzol to your sample. Note that the tissue is at the bottom of the tube, and sometimes along the side. Check that you can see some tissue (pink/gray color).

3.) To lyse the cells and homogenize: use glass homogenizer to crush sample to break up the tissue (be careful not to sqiuirt the sample all over yourself), then repetitively pipet the sample until the tissue is dispersed. The homogenate should be kept at room temperature for no longer than 15 minutes before centrifugation.

4.) Centrifuge the homogenate at full speed for 10 minutes in a Microcentrifuge .

REMEMBER TO BALANCE THE TUBES

5.) Transfer the supernatant to a new labeled 1.5 ml microcentrifuge

6.) Add 100 ul of absolute (100%) Ethanol. Invert the tubes gently and make sure the solution is well mixed. Keep at room temperature for 3-5 minutes.

7.) Centrifuge the solution at full speed for 5 minutes in a Microcentrifuge .

8.) Discard the supernatant. Make sure not to lose pellet.

9,) To wash the DNA: Add 500 ul of 75% Ethanol and invert the tube gently.

10.) Centrifuge the solution at full speed for 5 minutes in a Microcentrifuge .

11.) Discard the supernatant. Make sure not to lose pellet.

12.) To wash the DNA a second time: Add 500 ul of 75% Ethanol and invert the tube gently.

13.) Centrifuge the solution at full speed for 5 minutes in a Microcentrifuge .

14.) Discard the supernatant.

15.) Centrifuge the tubes again for 30 seconds in a Microcentrifuge ,and remove the residual Ethanol solution.

16.) Resuspend the DNA in 100 ul of water. Vortex the solution. Incubate at 42°C for 10 minutes and vortex again.

17.) Remove 10 ul of DNA solution and add 3 ul of 5X sample buffer.

18.) Run the 7 samples on a 1.2% Agarose Gel .

19.) Record data by Gel documentation.

REMEMBER TO SAVE YOU IMAGE AS A .TIF FILE (RAW DATA). AND MAKE SURE YOU HAVE SAVED IT TO THE RIGHT FOLDER.

PCR amplification of polymorphic markers

Set up the following two sets of PCR reactions:

Use a Master Mix based on 8 reactions. For groups of 3, you should do the Y-chromosome marker (Set 2) as a group.

Set 1

Component 1 2 3 4 5 6 7

Sample 1 DNA 5ul - - - - - -

Sample 2 DNA - 5ul - - - - -

Sample 3 DNA - - 5ul - - - -

Sample 4 DNA - - - 5ul - - -

Sample 5 DNA - - - - 5ul - -

Sample 6 DNA - - - - - 5ul -

Sample 7 DNA - - - - - - 5ul

Primer 3G5 FORWARD 1ul 1ul 1ul 1ul 1ul 1ul 1ul

Primer 3G5 REVERSE 1ul 1ul 1ul 1ul 1ul 1ul 1ul

10X Taq buffer 2.5 ul 2.5 ul 2.5 ul 2.5 ul 2.5 ul 2.5 ul 2.5 ul

50mM MgCl2 1ul 1ul 1ul 1ul 1ul 1ul 1ul

5mM dNTP 2ul 2ul 2ul 2ul 2ul 2ul 2ul

water 12.8ul 12.8ul 12.8ul 12.8ul 12.8ul 12.8ul 12.8ul

Taq DNA polymerase 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul

Total volume 25ul 25ul 25ul 25ul 25ul 25ul 25ul

Set 2

Component 1 2 3 4 5 6 7

Sample 1 DNA 5ul - - - - - -

Sample 2 DNA - 5ul - - - - -

Sample 3 DNA - - 5ul - - - -

Sample 4 DNA - - - 5ul - - -

Sample 5 DNA - - - - 5ul - -

Sample 6 DNA - - - - - 5ul -

Sample 7 DNA - - - - - - 5ul

Primer MUSY FORWARD 1ul 1ul 1ul 1ul 1ul 1ul 1ul

Primer MUSY REVERSE 1ul 1ul 1ul 1ul 1ul 1ul 1ul

10X Taq buffer 2.5 ul 2.5 ul 2.5 ul 2.5 ul 2.5 ul 2.5 ul 2.5 ul

50mM MgCl2 0.5ul 0.5ul 0.5ul 0.5ul 0.5ul 0.5ul 0.5ul

5mM dNTP 2ul 2ul 2ul 2ul 2ul 2ul 2ul

water 12.8ul 12.8ul 12.8ul 12.8ul 12.8ul 12.8ul 12.8ul

Taq DNA polymerase 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul

Total volume 25ul 25ul 25ul 25ul 25ul 25ul 25ul

PCR for 32 cycles: 94°C for 30sec, 57°C for 30 sec, 73°C for 45 sec.

Store samples in freezer.

Lab 5: Round 2 of PCR rections and polyacrylamide gel electrophoresis

Set up a 6% polyacrylamide gel. Polyacrylamide gel electrophoresis

Add 7 ul of 5X STOP loading dye to each PCR product, mix well, and load 10 ul onto the gel.

Load the first set of samples (lanes 1-7); then load the ladder (lane 8) and then the second set of samples (lanes 9-15)

Run the gel at 110 V for 50 min (or until blue dye is at bottom of gel) and stain the gel with Ethidium bromide for 10 minutes.

Record the data by Gel documentation

REMEMBER TO SAVE YOUR IMAGE AS A .TIF FILE (RAW DATA). AND MAKE SURE YOU HAVE SAVED IT TO THE RIGHT FOLDER.

Set up the following sets of PCR reactions:

Remember to set up a Master Mix.

Set 3

Component 1 2 3 4 5 6 7

Sample 1 DNA 5ul - - - - - -

Sample 2 DNA - 5ul - - - - -

Sample 3 DNA - - 5ul - - - -

Sample 4 DNA - - - 5ul - - -

Sample 5 DNA - - - - 5ul - -

Sample 6 DNA - - - - - 5ul -

Sample 7 DNA - - - - - - 5ul

Primer 6G2 FORWARD 1ul 1ul 1ul 1ul 1ul 1ul 1ul

Primer 6G2 REVERSE 1ul 1ul 1ul 1ul 1ul 1ul 1ul

10X Taq buffer 2.5 ul 2.5 ul 2.5 ul 2.5 ul 2.5 ul 2.5 ul 2.5 ul

50mM MgCl2 0.5ul 0.5ul 0.5ul 0.5ul 0.5ul 0.5ul 0.5ul

5mM dNTP 2ul 2ul 2ul 2ul 2ul 2ul 2ul

water 12.8ul 12.8ul 12.8ul 12.8ul 12.8ul 12.8ul 12.8ul

Taq DNA polymerase 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul

Total volume 25ul 25ul 25ul 25ul 25ul 25ul 25ul

Set 4

Component 1 2 3 4 5 6 7

Sample 1 DNA 5ul - - - - - -

Sample 2 DNA - 5ul - - - - -

Sample 3 DNA - - 5ul - - - -

Sample 4 DNA - - - 5ul - - -

Sample 5 DNA - - - - 5ul - -

Sample 6 DNA - - - - - 5ul -

Sample 7 DNA - - - - - - 5ul

Primer 5G4 FORWARD 1ul 1ul 1ul 1ul 1ul 1ul 1ul

Primer 5G4 REVERSE 1ul 1ul 1ul 1ul 1ul 1ul 1ul

10X Taq buffer 2.5 ul 2.5 ul 2.5 ul 2.5 ul 2.5 ul 2.5 ul 2.5 ul

50mM MgCl2 0.5ul 0.5ul 0.5ul 0.5ul 0.5ul 0.5ul 0.5ul

5mM dNTP 2ul 2ul 2ul 2ul 2ul 2ul 2ul

water 12.8ul 12.8ul 12.8ul 12.8ul 12.8ul 12.8ul 12.8ul

Taq DNA polymerase 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul 0.2ul

Total volume 25ul 25ul 25ul 25ul 25ul 25ul 25ul

PCR for 32 cycles: 94°C for 30secs 60 °C for 30 secs and 73°C for 45 secs.