Waksman Entrance Exam Flashcards
1
Q
Duckweed creates biofuel (alt. energy) by:
A
Converting starch to ethanol
2
Q
Duckweed degrades contaminants in the water which is an example of:
A
Bioremediation
3
Q
Polymer
A
A big molecule made up of smaller molecules that are linked together with similar bonds
4
Q
2 categories of super polymers:
A
Informational/Instructional
5
Q
DNA stands for:
A
deoxyribonucleic acid
6
Q
DNA is a double _____ and a polymer of _________
A
helix, nucleotides
7
Q
What makes up a nucleotide?
A
- 5 carbon sugar
- Phosphate group
- Nitrogenous Base (ATGC)
8
Q
Carbon makes _ bonds
A
4
9
Q
OH is also referred to as:
A
hydroxyl groups
10
Q
The phosphate group is linked to the 5’ carbon sugar at
A
5’
11
Q
Where do nucleotides join
A
5’ and 3’
12
Q
Ribose is found in
A
RNA
13
Q
Deoxyribose is found in
A
DNA
14
Q
Which lacks a hydroxyl group at the 2’ carbon
A
Deoxyribose
15
Q
Oxygen’s of the phosphate group are negatively charged, therefore:
A
DNA is negatively charged
16
Q
Oxygen makes _ bonds
A
2
17
Q
If the oxygen does not make 2 bonds, it becomes _
A
negative
18
Q
A is
A
Adenine
19
Q
T is
A
Thymine
20
Q
C is
A
Cytosine
21
Q
G is
A
Guanine
22
Q
Purines have how many rings
A
2
23
Q
Which are purines
A
A and G
24
Q
Pyrimidines have how many rings
A
1
25
Which are pyrimidines
T and C
26
Which bond through 2 hydrogen bonds
A and T
27
Which bond through 3 hydrogen bonds
G and C
28
DNA is anti
parallel
29
The complimentary strand of 5' to 3' is
3' to 5'
30
Polynucleotide chains are
A chain of the molecules of the 4 bases
31
How does a polynucleotide chain form?
The O of the 3' hydroxyl group on the chain attacks the phosphate of a nucleotide tri-phosphate, eliminating the 2 outermost phosphates and releasing H20
32
Genomic DNA library
Contains ALL sequenced DNA of an organism, includes both coding and non-coding regions
33
cDNA DNA library
Contains mRNA - ONLY the DNA that can be used to makee proteins, and includes ONLY coding regions
34
Generic process
cloning and overnight cultures
35
DNA fragments must be _______ and ______ to be studied
amplified and purified
36
Can you purify it alone
No, its almost impossible
37
Fragments must be inserted (cloned) intro vectors
Circular DNA molecules that replicate inside bacterium
38
What vector does Waksman use?
pTriplEx2
39
What are the 3 steps of the cloning process
1. Digest
2. Ligate
3. Amplify`
40
What does digest mean?
Insert and vector are cut/digested at sites A and B by enzyme Sfil
41
What does ligate mean?
Insert and vector are joined (ligated) at sites A and B
42
How do you amplify the plasmid?
Plasmid is put in bacterium for amplification
43
What is ON
Overnight cultures (bacterium transformation)
44
What is step 1 of cloning
Make cells competent with CaCl2 (Calcium Chloride), so that the cells and plasmids are both negatively charged.
45
What is step 2 of cloning
Transform plasmid DNA - some cells will take up the plasmid
46
What is step 3 of cloning
Plasmid DNA replication - bacterium naturally replicates plasmid
47
What is step 4 of cloning
Eliminate cells without plasmid - cells without plasmid die in antibiotics (ampicillin)
48
What is step 5 of cloning
Cell replicates - Bacterium cells replicate naturally with plasmid
49
What is step 6 of cloning
Plate the cells - replicates cells become colonies
50
The pTriplEx2 vector contains a
selectable marker
51
What initiates replication?
Origin of replication
52
What does the Ampicillin selectable resistance marker (AmpR) do?
Ensures only bacteria containing plasmid will replicates and grow.
53
What does the lacZ gene do?
Indicates which colonies and plasmids have the insert.
54
What enzyme does the lacZ gene make?
B-galactosidase
55
What substance does B-galactosidase react with to create blue-colored colonies?
X-gal
56
What can happen if the lacZ gene is disrupted by a DNA insert?
It will no longer produce the blue color. Instead, colonies with the DNA insert will be white
57
Which type of colony should we choose?
The ISOLATED WHITE colony
58
Why do we overnight cultures?
to grow bacteria to prepare the DNA for PCR and RD.
59
What is step 1 of ON?
Nourish - Grow the culture in Luria broth with ampicillin.
60
What is step 2 of ON?
Shake - Tap or vortex the tube to help the growth.
61
What is step 3 of ON?
Incubate - Leave the culture overnight at 37°C.
62
Darker yellow color means the ON is fully grown or not yet fully grown?
Fully grown
63
How do you name your ON on the clone sheet?
(School number)(Initials CAPS)(Clone#).(Year) - ex. 20RN3.24
64
What does PCR stand for?
Polymerase Chain Reaction
65
PCR rapidly produces billions of copies
Yes
66
What is step 1 of PCR prep?
Dilute the ON in water -Undiluted cells do not work properly in PCR → causes unclear PCR band.
67
What happens if we use too much DNA?
The primers and nucleotides will get used up too fast
68
What is step 2 of PCR prep?
Prepare the 5X mix
69
What is the 5X Mix made up of?
- Use sterile distilled water (ddH₂O) to control the volume and concentration of the reagents.
- Use primers - SP and XP primers attach to the plasmid DNA.
- 2X Taq Mix - This mix contains Taq buffer, dNTPs, and Taq polymerase.
70
The 5X Mix is used for how many clones (PCR tubes)
4
71
What is step 3 of PCR prep?
The 5X Mix (23ul each 4x) and the diluted cultured (2ul each 4x) are added together.
72
What is step 4 of PCR prep?
Place DNA in thermal cycler
73
What is step 1 of the PCR process?
Initial Denaturation: Heat the DNA to 94C for 5min - this high temp. breaks apart the double-stranded DNA into two single strands by breaking the weak hydrogen bonds holding them together
74
What is step 2 of the PCR process?
Amplification: (Repeated 30x):
a. 94C for 1min - re-denaturation
b. 50C for 1min - annealing, primer bind to the single stranded DNA to prepare for copying.
c. 72C for 1min - synthesis, Taq enzyme adds nucleotides to form new DNA strands, making copies.
75
What is step 3 of the PCR process?
Additional Elongation: 72C for 5min to make sure that any remaining DNA strands are fully extended.
76
What is step 4 of the PCR program?
End Program: Cooled down to 4C to keep samples stable until taken out.
77
What does gel electrophoresis do
To determine the size of DNA fragments
78
What is step 5 of PCR?
Gel electrophoresis
79
How does DNA move in the gel electrophoresis?
1. Load the DNA in the wells at the negative end of the gel
2. Since DNA is negative, it will migrate to the positive end
3. smaller DNA fragments move further than the larger fragments, causing the fragments to be separated by size.
80
What's a messy band
If the gel has unclear or smeared bands, it may be due to random priming. This happens when primers bind to unintended spots of the DNA.
81
Why could there be multiple bands?
- The primers are binding to multiple places on the DNA
- There are two different plasmids or colonies present.
82
Why could there not be any bands on the PCR
The inserted DNA is too large to be properly amplified.
83
What is an enzyme?
A protein that breaks things down
84
How do we split the ON between PCR and RD
Half and half
85
How do you find the size of the insert?
compare the DNA fragment to the marker lane
86
The uncut lane is the
control group
87
What does the sample contain that makes it visible under a UV light?
ethidium bromide
88
The gel is submerged in _____, which carries the current throughout the gel
buffer
89
What does a bright band with a lot of background mean?
too much DNA (overloaded)
90
What do faint bands mean?
too little DNA
91
What does it mean when there are no bands except for the DNA ladder?
no ethidium / no samples / no cells
92
Restriction enzymes are:
cleave DNA at a specific nucleotide sequence
93
PvuII enzyme cleaves the vector at how many sites?
2
94
Two fragments are formed from the cleavage of the PvuII enzyme . What are they?
vector backbone and the insert
95
pTriplEx2 enzyme is made up of how many PvuII enzymes?
2
96
What does it mean if there are 3 bands in the cut lane?
Too large of an insert
97
What are the three different shapes of tangled DNA?
Top - open circle
Middle - Linear
Lowest - supercoil
98
Which tangled shape of DNA will migrate fastest and why?
Supercoil, it is the thickest.
99
What does N/A mean?
the sample is contaminated
100
What does Contamination mean
Some kind of external DNA got into the tested DNA
101
Each sequence is in _ places on the vector
2
102
Do you calculate the insert size from the cut or uncut lane?
cut
103
How do you calculate insert size with 2 bands?
Lowest bad - 700bps
104
How do you calculate insert size with 3 bands?
Sum of lower two bands - 700bps
