1. In Lab Today:
1. Finish Lab 1 looking at slides of bacteria and pond water
2. Prepare for the sequencing of our mtDNA (Lab 2)

2. What do all of these have in common?

3. Cells All living things are made of cells Two types of cells
Prokaryotic
Eukaryotic

4. What are the differences between prokaryotic and eukaryotic cells?
Prokaryotic cells lack a nucleus and membrane bound organelles
Eukaryotic cells have a nucleus, more specialized, have membrane bound organelles

5. What kind of cell is this?

6. What kind of cell is this?

7. What kind of cell is this?

8. mtDNA Sequencing: DNA Replication

9. The Polymerase Chain Reaction (PCR)

10. Mitochondria

11. Maternal Inheritance

12. Our Protocol: mtDNA Sequencing
Two Steps:
Extract mtDNA
Amplify this DNA (PCR!)

13. Our Protocol: mtDNA Sequencing
Extracting the DNA:
Swish the saline solution to loosen cells (>10 seconds, minimal eating, esp. fruit!)
Spin the cells (10 min). Pour off the liquid.
Mix the Chelax Resin and add it to your cell pellet.
Transfer 500 ul of this solution to a clean tube, mixing carefully!

14. Our Protocol: mtDNA Sequencing
Extracting the DNA:
Cap your tube tightly and place in the boiling water bath for 10 minutes.
Allow the tube to cool and then spin for 30 seconds.
Carefully transfer 200 uL of the liquid to a clean tube, avoiding the pellet. This is your DNA solution!

15. Our Protocol: mtDNA Sequencing
Preparing the PCR Reaction:
Obtain a PCR tube with bead and 225uL of red solution from instructor. Handle this tube gently and record the number written on it!
Carefully add 2.5 uL of your DNA prep to the red PCR solution.
Cap the tube tightly and place it in the thermocycler.

16. I will check your PCR reaction and send your DNA to be sequenced!
What Next?
I will check your PCR reaction and send your DNA to be sequenced!