MB311 Midterm Exam
Name ______
Question 1 (75 points)
The DNA synthesis facility sends you a 20-nucleotide primer as a dry powder; the molecular weight of this oligonucleotide is 6600. You dissolve the oligonucleotide in 1 ml of sterile distilled water; this is your "concentrated primer stock." You mix 5 l of the "concentrated primer stock" with 495 l of water and measure the absorbance at 260 nm; the reading is 1.5. A spectrophotometer reading of 1 absorbance (OD) unit at 260 nm indicates a concentration of 33 g/ml for a short single-stranded oligonucleotide.
What is the concentration of the "concentrated primer stock"?
4950 g/ml or 750 M
______
Please show your calculations. You may express the answer as g/ml, or you may give the
micromolar (M) concentration.
1M = 6600 g/l; 1 M = 6600 g/l = 6.6 g/ml; 10 M = 66 g/ml
5/500 = 1/100 dilution
1.5 OD units x 33 g/ml / OD unit = 49.5 g/ml in cuvette;
stock = 49.5 g/ml x 100 = 4950 g/ml
stock = 4950 g/ml x 1 M/6.6 g/ml = 750 M
How much must you dilute the "concentrated primer stock" to make a 10 M solution for use in PCR?
1/75
______
Please show your calculations.
66 g/ml / 4950 g/ml = 1/75 = 0.0133 or 10 M / 750 M = 1/75
Name______
Question 2 (75 points)
The "27F" PCR primer you used in Experiment 3 had the following sequence:
5'- AGA GTT TGA TCC TGG CTC AG -3'
Assume this primer was 100% complementary to the template DNA, and your PCR reaction contained 100 mM NaCl. What was the melting temperature (Tm) of the duplex DNA formed between this primer oligonucleotide and the template DNA under these conditions?
Tm = 16.6 log[Na] + 0.41 (% G+C) + 81.5 - 500/bp, where % G+C = percentage expressed as a whole number (for example, "50", not "0.5", indicates 50%), [Na] = molar salt concentration, and bp indicates length of DNA:DNA hybrid in base pairs.
27F primer = 20 bases; 50% G+C (10 G+C/20 total)
100 mM NaCl = 0.1 M NaCl
Tm = 16.6 log (0.1) + 0.41 (50) + 81.5 - 500/20
Tm = 16.1 (-1) + 0.41 (50) + 81.5 -25
Tm = -16.6 + 20.5 +81.5 -25 = 60.4 oC
Name ______
Question 3 (50 points)
"Ap" indicates the beta-lactamase (bla) gene, which encodes resistance to ampicillin. "Tet" indicates the tetA gene, which encodes resistance to tetracycline.
You did the molecular cloning experiment shown in the figure, and you transformed the DNA into competent E. coli (strain DH5), which is sensitive to ampicillin and tetracycline. You spread the transformed cells on LB agar plates that contain an antibiotic to kill nontransformed bacteria. What antibiotic did you put in the agar? Why?
Ampicillin. The tetA gene is disrupted by the lux operon.
You also plated competent cells that did not receive DNA. Explain why.
This was a negative control to ensure that the competent cells were not already resistant to ampicillin. It also tests whether the medium contained sufficient ampicillin to kill untransformed host cells.
Name______
Question 4 (32 points)
Supply preferred words for the following jargon:
Jargon:Preferred Terms:
a considerable number of many
a decreased amount of less
a majority of most
a great deal of much
absolutely essentialessential
accounted for by the fact thatbecause
count the number of count
employuse
for the purpose of for
is defined asis
is similar toresembles
it has been shown that(omit and cite reference)
prior tobefore
red in colorred
the vast majority ofmost
utilizeuse
Name______
Question 5. (25 points)
Define IMRAD.
Introduction, Methods, Results, and Discussion.
What is wrong with this title? “Action of Antibiotics on Bacteria”
This title contains general rather than specific terms.
“Action of Streptomycin on Mycobacterium tuberculosis” is better.
What is one rule for a properly written materials and methods section of a paper?
The materials and methods section must provide enough information to allow colleaguesto repeat the experiments.
Which voice is usually best, active or passive?
Active voice.
What tense should you use to state previously published findings?
Present tense.
When you describe your work in a paper, what tense should you use?
Past tense.
Which sentence is best?
“There is another method that is gaining acceptance.”
“Another method is gaining acceptance.”
The shorter sentence is best.
Name______
Question 6. (18 points)
The authors of the paper entitled "Phosphate Starvation Response of lux-Tagged Pseudomonas fluorescens Reporter Bacteria in the Barley Rhizosphere" used gnotobiotic soil for some of their experiments. Define "gnotobiotic."
gno-to-bi-o-sis: 1. the study of organisms or conditions that are free of germs or contaminants or to which a known germ or contaminant has been introduced for purposes of study.
Derived word: gno to-bi-ot'ic, adj
Question 7. (25 points)
Will a particular primer:template duplex DNA be more stable in 10 mM NaCl or 100 mM NaCl?
In other words, will the melting temperature (Tm) of double-stranded DNA be higher in 10 mM NaCl or 100 mM NaCl?
The Tm will be higher in 100 mM NaCl.
Question 8. (50 points)
The DNA synthesis facility sends you a 20-nucleotide primer as a dry powder; the data sheet indicates that the tube contains 50 nmol of oligonucleotide. You want to prepare a 100 M stock. What volume of buffer should you use to dissolve the oligonucleotide? Please show your calculations.
500 l
50 nmol/500 l = 0.1 nmol/l = 100 nmol/ml = 100 mol/l = 100 M
Name______
Question 9. (50 points)
You have an 8 kb plasmid vector with a single EcoRI site (circle; see figure). You digested the plasmid with EcoRI and ligated the plasmid to a 4 kb EcoRI restriction fragment (bold line), forming a 12 kb recombinant plasmid (not shown). The plasmid has a promoter (P + arrow) that drives transcription clockwise through the EcoRI site. The 4 kb EcoRI fragment contains an open reading frame (bold arrow labeled ORF) that you want to express from this promoter. The EcoRI fragment could have inserted into the vector plasmid in either orientation relative to the promoter. What restriction enzymes do you need to determine the orientation of the 4 kb fragment relative to the promoter? Do the minimum number of digestions required to determine the orientation. What size restriction fragments do you expect for the desired orientation?
BamHI produces fragments of 2, 4, and 6 kb from the desired plasmid; the opposite orientation yields BamHIfragments of 2, 2, and 8 kb. Any other enzyme gives the same pattern for both orientations.