Biotechnology Unit
Science Department
Ms. Martinez
2009
1. What was the purpose of this
lab?
1. What was the purpose of this
lab?
Have
a hands-on experience using tools
and techniques commonly used molecular
biology
To introduce some of the volumetric
measurements used in molecular biology
2. In a molecular biology lab,
what units are solutions
measured out in?
2. In a molecular biology lab,
what units are solutions
measured out in?
microliters (µL)
3. Why do molecular biologists
use such small volumes
amounts of materials?
3. Why do molecular biologists
use such small volumes
amounts of materials?
-Cost $$ (these materials are
expensive and the difficult to obtain)
4. Why are micropipettors
used?
4. Why are micropipettors
used?
To aliquot small amounts of
liquids
5. What are 4 precautions when
using the micropipettor?
5. What are 4 precautions when
using the micropipettor?
1.Do
not set the adjustment below 2 µL or
above 20 µL
2.Always use a fresh tip
3. Do not lay down a micropipette with fluid
in the tip or hold it with the tip pointed
upward
4. Avoid “snapping” the plunger
6. When working with the
micropipettor, what should
the display window read for
20.0 uL?
6. When working with the
micropipettor, what should
the display window read for
20.0 uL?
2
0
0
7. When working with the
micropipettor, what should
the display window read for
12.4 uL?
7. When working with the
micropipettor, what should
the display window read for
12.4 uL?
1
2
4
8. How many stops are on the
plunger of the micropipettor?
8. How many stops are on the
plunger of the micropipettor?
2
9. What is the function of the
stops on the micropipettor?
9. What is the function of the
stops on the micropipettor?
-1st stop—dispense liquid
-2nd stop--pushes
any remaining
-fluid out
10. What does aliquot mean?
10. What does aliquot mean?
To transfer a sample of some part
of a whole
11. What is a locus?
11. What is a locus?
-A place or location on a
chromosome
(e.g., Alu+, Alu- in the tPA
intron).
12. From what cells was your
DNA collected?
12. From what cells was your
DNA collected?
Buccal Epithelial Cells
13. Why was it important that
the cells you collect from your
mouth are mixed with
Chelex?
13. Why was it important that
the cells you collect from your
mouth are mixed with
Chelex?
1.
Attract metals
2. Lyse cells
14. Identify 2 reasons why one
might have metal in their
mouth.
14. Identify 2 reasons why one
might have metal in their
mouth.
1.
Dental Work
2. Food
15. What is the purpose of
boiling the cell sample?
15. What is the purpose of
boiling the cell sample?
To lyse the cell to liberate the
DNA
16. What does the word lyse
mean?
16. What does the word lyse
mean?
To break apart
17. What does the term to
liberate DNA mean?
17. What does the term to
liberate DNA mean?
Set free
18. What enzyme was used in
this lab?
18. What enzyme was used in
this lab?
Taq polymerase
(isolated from Thermus aquaticus)
19. What do enzymes do to all
reactions? How?
19. What do enzymes do to all
reactions? How?
They speed up reactions by
LOWERING activation energy.
20. What is a quick and easy way
to separate genomic DNA
from the cell debris?
20. What is a quick and easy
way to separate genomic
DNA from the cell debris?
Microcentrifuge
Machine
21. What is PCR is an
abbreviation for?
21. What is PCR is an
abbreviation for?
Polymerase Chain Reaction
22. What does amplify mean?
22. What does amplify mean?
To make more copies
23. LIST
2 examples of PCR
usages.
23. LIST
1.
2 examples of PCR
usages.
to amplify DNA evidence left at a
crime scene.
2. Paternity Tests
24. How many times does the
cycle of denaturing occur?
24. How many times
does the cycle of denaturing occur?
94 C: Denature DNA
56 C: Anneal Primers to Template
72 C: Activates Taq Polymerase
Repeats 31 times
25. How many copies of DNA will
be made? in how many
hours?
25. How many copies of DNA will
be made? in how many
hours?
1 Billion
3-4 hours
26. What is the name of the
machine where PCR occurs?
26. What is the name of the
machine where PCR occurs?
Thermal Cycler
27. Describe how the Thermal
Cycler works.
27. Describe how the Thermal
Cycler works.
-Heat (94oC) to denature DNA strands
-Cool (56oC) to anneal primers to template
-Warm (72oC) to activate Taq polymerase,
which extends primers and replicates DNA
28. Identify the locus we
amplified.
28. Identify the locus we
amplified.
tPA
The region we amplified is located in
an intron (non-translated region), of
the tPA gene.
29. Which chromosome number
is the locus we amplified
carried on?
29. Which chromosome
number is the locus we
amplified carried on?
Water
30. What does the tPA gene
code for?
30. What does the tPA gene
code for?
NOTHING!
It is an intron
31. What is an intron?
31. What is an intron?
non-translated region of DNA
32. What does dimorphic
mean?
32. What does
dimorphic mean?
Chromosome 8
tPA gene
3’
5’
e
i
e
i
e
i
5’
e
3’
300bp
primer
Alu element
primer
400bp
primer
primer
100bp
The locus has two
forms
(ALU+ and ALU-)
33. How many bp’s long is the
ALU+ allele?
33. How
Chromosome 8
many bp’s long is the
ALU+ allele?
400 bp
tPA gene
3’
5’
e
i
e
i
e
i
5’
e
3’
300bp
primer
Alu element
primer
400bp
primer
primer
100bp
34. How many bp’s long is the
ALU- allele?
34. How many bp’s
Chromosome 8
long is the
ALU- allele?
tPA gene
3’
5’
e
i
e
i
e
i
5’
e
3’
300bp
primer
Alu element
primer
400bp
primer
primer
100bp
100 bp
35. What are ALU elements?
35. What are ALU elements?
-Random pieces of DNA
-They don’t have any function
in the human genome.
36. How long are ALU
elements?
36. How long are
ALU elements?
Chromosome 8
300 bp
tPA gene
3’
5’
e
i
e
i
e
i
5’
e
3’
300bp
primer
Alu element
primer
400bp
primer
primer
100bp
37. How many copies of ALU
do we carry?
37. How many copies of ALU
do we carry?
Over 1,000,000
38. What is amplification of
DNA by PCR dependent
on?
38. What is amplification of
DNA by PCR
dependent on?
Chromosome 8
tPA gene
3’
5’
e
i
e
i
e
i
5’
e
3’
300bp
primer
Alu element
primer
400bp
primer
primer
100bp
Primers that target
tPA
39. What are primers?
39. What
are primers?
-Short, preexisting DNA nucleotides to
which new DNA nucleotides can be added
-(Two different primers are used to
target the tPA locus amplified by PCR).
40. What reagents were part
of Master Mix I?
40. What reagents were part
of Master Mix I?
-Two primers that target the tPA locus
-ATP
-Buffer
-Taq polymerase, an enzyme from the
bacteriumThermus aquaticus
-very pure water
41. What was the purpose of
a buffer?
41. What was the purpose of a
buffer?
To resist change in pH
42. What was the purpose of
ATP?
42. What was the purpose of
ATP?
Energy
43. What
reagent was part of
Master Mix II?
43. What
reagent was part of
Master Mix II?
-MgCl2 (enzyme co-factor)
44. What was the purpose of
Magnesium Chloride, MgCl2?
44. What was the purpose of
Magnesium Chloride?
Enzyme co-factor (activates MMI)
45. What is gel
electrophoresis?
45. What is gel electrophoresis?
Technique to separate DNA
fragments by using electricity
MMI, MMII
46. What is the charge on a
DNA molecule?
46. What is the charge on a
DNA molecule?
Negative
47. Why does DNA travel to
the positive end of the gel?
47. Why does DNA travel to
the positive end of the gel?
Opposite charges attract (DNA is
negative)
48. How
does a person obtain
2 copies of the ALU+
element?
48. How
does a person obtain
2 copies of the ALU+
element?
They receive one copy from mom
AND one copy from Dad
49. How does a person obtain 1
copy of the ALU+ element?
49. How does a person obtain
1 copy of the ALU+
element?
They receive one copy from
mom OR one copy from Dad
50. How does a person obtain 0
copies of the ALU+ element?
50. How does a person obtain
0 copies of the ALU+
element?
They DID NOT receive one copy
from mom OR one copy from Dad
51. What are transgenic
organisms?
51. What are
transgenic
organisms?
-Organisms that have
DNA from other
organisms
Transgenic Tobacco Plant
-ex Transgenic
bacteria have the human
T
insulin gene to produce synthetic
human
s
insulin
52. How
do you make a
transgenic organism?
52. How
do you make a
transgenic organism?
Genetic Engineering
53. What
is a clone?
53. What
is a clone?
A member of a population of
genetically identical cells produced
from a single cell.
54. What 2 mammals have
been cloned?
54. What 2 mammals have
been cloned?
Sheep (Dolly 1st), cows pigs mice
pandas.
Study!