1

Name

MODEL ANSWERS

ID number

MASSEY UNIVERSITY
ALBANY CAMPUS
LABORATORY TEST FOR

203.300 DNA TECHNOLOGY

Semester 1, 2010

Time allowed: ONE HOUR

There are FOUR questions in this test.
Answer all questions.
All questions should be answered on these question sheets.
Write your name and ID number in the spaces provided above.
All questions are of equal value (10 marks each).
Allow approximately fifteen minutes to answer each question.
This test is worth 10% of your total assessment for this paper.
Your lab manual, lab notebook and a non-programmable calculator are permitted.
Question 1

Calculations

Questions 2 and 3

Calculations and Understanding Methods

Question 4

Analysis and Interpretation

For examiners use only

1

Total

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Question 1
a)

How would you make 250 ml of the following buffer?

25 mM Tris, 1.5 mM EDTA, 5% (w/v) sucrose, and 10% (v/v) glycerol
(MWTris = 121.1 g/mol; MWEDTA = 292.3 g/mol)

(5 marks)

Amount of Tris required, n = c.V

= (25 x 10-3 M) x 0.25 L
= 0.00625 mol
Mass of Tris to weigh out, m= n.Mr
= 0.00625 mol x 121.1 g/mol
= 0.76 g
Repeating for EDTA gives m = 0.11 g
Sucrose at 5% (w/v): 5% of 250 mL is 12.5 mL, but its weight per volume, so weigh out 12.5 g.
Glycerol at 10% (v/v): 25 mL in the final volume of 250 mL.
Water: make the buffer up to a final volume of 250 mL.

b)

A 25-fold dilution of your pET14b plasmid DNA stock gives an absorbance reading of
0.14 at 260 nm. Using the rule that an A260 of 1.0 indicates 50 g/mL of doublestranded DNA, determine the concentration of the plasmid DNA stock in ng/L.
(3 marks)
A260 (undiluted)

= 0.14 x 25
= 3.5

A260 of 1 corresponds to c = 50 g/mL

Therefore, A260 of 3.5 corresponds to c = 3.5 x 50 g/mL
= 175 g/mL
= 175 ng/L

c)

You wish to use 10 ng of your plasmid stock from (b) above to transform E. coli by
electroporation. How would you accurately dispense 10 ng into the tube containing
your electrocompetent cells?
(2 marks)
Make a sensible dilution, so that you do not have to pipette a fraction of a L. For example;
Use the P2 (or P10) pipettor to add 1 L DNA to 16.5 L H2O. The resulting solution is 10 ng/L.
Pipette 1 L of this into the tube containing your cells.

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Question 2
You are attempting to sub-clone the metC gene into a protein expression vector called
pCA24N. Fortunately, one of your co-workers has already cloned metC into the pCR8
cloning vector.
a)

b)

You digest 4.0 g of pCR8-metC (4,002 bp) with NdeI and BamHI, in order to release
the 1,185 bp metC gene. Your gel purification protocol is 50% efficient. How much of
the purified metC fragment do you end up with?
(2 marks)
Maximum yield of metC

= (1185/4002) x 4.0 g
= 1.18 g

Actual yield

= max. yield x 0.5

= 0.59 g

You plan to ligate the 1,185 bp fragment from (a) above to 150 ng of pCA24N
(5,332 bp) that has also been digested with NdeI and BamHI. If you wanted to add
equal numbers of vector and insert molecules to the ligation reaction, how many
nanograms of insert DNA should you use?
(2 marks)
150 ng x (1185 bp/5332 bp) = 33 ng

c)

You set up your ligation reaction, and incubate it overnight at room temperature.
Next, you decide to desalt the DNA with a Micro Bio-Spin column, before
transforming E. coli. Describe the principle by which the Micro Bio-Spin column
separates the DNA from the other reaction components (ligase enzyme, buffer salts,
ATP etc.). Use a diagram if necessary.
(2 marks)
Gel filtration (also known as size exclusion): the column contains porous beads. Large molecules (i.e.
DNA) cannot enter the pores, so elute rapidly from the column. Small molecules (ligase, salts, ATP
etc.) enter the pores and are therefore retained.

d)

You use your desalted DNA to transform an aliquot of E. coli cells by electroporation.
The time constant is 0.2 ms, and the next day you have no colonies on your plates.
What is the most likely explanation for your lack of colonies?
(1 mark)
The time constant is very low: the current was conducted by salts, rather than E. coli cells. This
suggests either that the desalting step failed, or that the cells had not been washed extensively enough
during their preparation.

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e)

You decide to make a fresh batch of electrocompetent cells, in order to repeat your
experiment. This time, before using them with your ligation products, you transform
an aliquot with 1.0 ng of pUC19 plasmid. After electroporation, you add SOC broth to
a total volume of 500 L, then spread 100 L of a 104-fold dilution on an LB+Amp
plate. After incubation, you count 94 colonies on the plate. Calculate the number of
transformants per g of DNA.
(3 marks)
Colonies from the electroporation

= 94 x 104 x (500 L/100 L)

= 4.7 x 106

This was from 1.0 ng, therefore the transformation efficiency per g is:
Transformation efficiency

= 4.7 x 106 x (1 g/1 ng)

= 4.7 x 109 transformants per g

Question 3
Your new transformation from Question 2 appears to have worked perfectly. You pick a
colony from the vector + insert ligation plate and purified the recombinant plasmid. Now,
you plan to order two universal primers, to test whether the plasmid contains the desired
metC gene insert.
a) What is meant by the term universal primers?
(1 mark)
Short pieces of single-stranded DNA that anneal to commonly used sequences in standard cloning
vectors (such as the T7 forward and reverse sequences). Being vector-specific, they allow amplification
or sequencing of any insert in a given vector.

You order primers with the following sequences:

b)

FORWARD:

5 GAT AAC AAT TTC ACA CAG AAT TCA TTA G 3

REVERSE:

5 CCC ATT AAC ATC ACC ATC TAA TTC 3

Calculate the Tm values of the two primers shown above.

Tm

(2 marks)

= 2(#A + #T) + 4(#G + #C)

Tm (for)

72C

Tm (rev)

66C

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c)

The next step is to set up a PCR with these two primers. Complete the following
table for preparing your reaction:
Component (and
stock concentration)

Final amount or
concentration required

Volume to add

1x

4 L

dNTP mix (2 mM)

0.3 mM

6 L

Forward primer
(10 M)

0.25 M

1 L

Reverse primer
(10 M)

0.25 M

1 L

Taq polymerase
(5 units/L)

2 units

0.4 L

Template DNA
(10 ng/L)

20 ng

2 L

To final volume of 40 L

25.6 L

Taq buffer (10x)

Water

(5 marks)

d)

Describe two control reactions that you should also carry out when you do this PCR.
(2 marks)
Negative control: water instead of template DNA.
Positive control: purified vector DNA (with stuffer fragment).

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Question 4
a)

The figure below is of agarose gel electrophoresis of double-stranded, linear DNA

stained with ethidium bromide.
Lane 1
1 Kb Plus ladder (sizes shown in kb).
Lanes 2-5 DNA quantitation standards with the amounts indicated applied to the gel.
Lane 6
A 2 L aliquot of a gel-purified DNA fragment to be quantitated (mixed with
3 L water and 1 l loading dye, and the entire mixture run on the gel).

Lanes

(i) Estimate the approximate amount of DNA (in nanograms) in 1 l of the solution of
gel-purified DNA fragment.
2 L aliquot looks to have approx. 75 ng, therefore 1 L 37.5 ng
(1 mark)
(ii) Estimate the approximate size of the gel-purified DNA fragment.
From the ladder, approx. 3.2 kb

(1 mark)

(iii) Using your answers from parts (i) and (ii), calculate the concentration of the DNA
fragment in nmol/l. (The approximate molecular mass of a single nucleotide is
330 g/mol)
(4 marks)
MW of DNA fragment

= 3,200 bp x 2 nt/bp x 330 g/mol/nt

= 2,112,000 g/mol

cDNA = (37.5 x 10-9 g/L) / (2,112,000 g/mol)

= 1.78 x 10-14 mol/L
= 1.78 x 10-5 nmol/L

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