Download mtDNA Lab2008

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)
What do all of these have in
common?
Cells


All living things are made of cells
Two types of cells
Prokaryotic
 Eukaryotic

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
What kind of cell is this?
What kind of cell is this?
What kind of cell is this?
mtDNA
Sequencing:
DNA
Replication
The
Polymerase
Chain
Reaction
(PCR)
Mitochondria
Maternal
Inheritance
Our Protocol: mtDNA Sequencing
Two Steps:
1.
2.
Extract mtDNA
Amplify this DNA (PCR!)
Our Protocol: mtDNA Sequencing
Extracting the DNA:
1.
2.
3.
4.
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!
Our Protocol: mtDNA Sequencing
Extracting the DNA:
5.
6.
7.
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!
Our Protocol: mtDNA Sequencing
Preparing the PCR Reaction:
1.
Obtain a PCR tube with bead and 225uL of red
solution from instructor. Handle this tube
gently and record the number written on it!
1.
Carefully add 2.5 uL of your DNA prep to the
red PCR solution.
Cap the tube tightly and place it in the
thermocycler.
2.
What Next?
I will check your PCR
reaction and send
your DNA to be
sequenced!
http://www.geneticorigins.org