In this document you can find several practice test questions.

Questions will be a combination of theory courses, practice lab and bioinformatics and tutor.
These are a number of example questions, test questions can also include other topics such as:
restriction enzyme analysis, isolation of other type of DNA or protein, analysis of DNA / protein
(agarose gel / SDS-Page), protein analysis.

Plasmid DNA isolation 15 points (1,1,1,1,6,3,2)

In the lab, you received two strains of bacteria from a colleague: one has the plasmid that codes for
mCherry and one has the plasmid that codes for tdTomato. To make sure that nothing has been
swapped, you want to confirm that the two bacterial strains do indeed have these plasmids.

XhoI
XhoI
p-mCherry p-tdTomato 2810
8172 bp 2810
8892 bp
EcoRI
EcoRI
EcoRI EcoRI
5590
3289
4870 3289

1. A plasmid consist of DNA. A piece is shown in the figure below. Indicate in the empty boxes
whether it is a 3' or 5' end.

5’

3’

3’

5’
 2. Bacteria multiply every 20 minutes under ideal conditions. Replication starts at the Origin of
Replication (ORI). Part of the replication is shown schematically below. Put the correct
concepts in the empty boxes.

RNA primer
Helicase

Leading
DNA polymerase (III)

Lagging strand of Okazaki fragment

3. Name 3 differences between DNA and RNA.

Answer:
Dubbel stranded vs single stranded,
Ribose vs deoxyribose
RNA contains uracil and DNA thymine as nitrogen base

4. For isolating the DNA from the two E.coli strains, you have found 2 protocols in the lab. You will
find two protocols for DNA isolation in your documentation (see appendix II of this document).
Unfortunately, it does not say for which cells and what type of DNA these protocols are suitable.

a) Which of the two protocols, A or B, is suitable for plasmid DNA isolation from E.coli?
Answer:
Protocol B is suitable for plasmid isolation
b) Explain your answer in a) by explaining the main steps of both protocols and why plasmid DNA
can/cannot be isolated.
Answer:
Protocol A:
- breaking cell wall with lysozyme
- addition of RNase, SDS, proteinase K to degrade RNA and protein (and SDS dentatures
protein)
- chloroform/isoamylalkohol: separation of DNA, proteins and lipids: DNA in water phase,
proteins in interphase, lipids and proteins in chloroform/isoamylalkohol
- addition of ethanol: precipitation of DNA
Therefore: no separation of chromosomal DNA and plasmid DNA

Protocol B:
- addition of SDS and NaOH (very high pH) breaking cell wall, denaturation of proteins and
DNA
- RNA is degraded because RNA is present
- addition of KAc (pH 4.8): renaturation of reversibly denatured structures: only plasmid DNA
can renature because it is small and the two DNA rings are still attached to each other,
chromosomal DNA is too large
- use supernatant (renatured plasmid) to precipitate DNA with isopropanol
Therefore: isolation of only plasmid DNA

5. You want to isolate at least 10µg of plasmid DNA from each of the two strains of bacteria.
How much overnight culture do you use? (Keep in mind that you lose up to 50% plasmid DNA
during isolation.)
You have the following information:
• Each bacterial cell has 1000 plasmid molecules
• The size of the plasmid is around 8000 bp
• Avogadro's constant is: 6.02*10 23
• The average mole mass of a base pair (AT or GC) is 660 g/mol.
• You can assume that the optical density (OD at 600nm) of the overnight culture is 1 and
this corresponds to 6*108 cells/mL.
 Answer:
In 1 ml are 6*108 cells, and 6*1011 plasmid molecules (number of cells * number of
plasmid molecules per cell).
These are 6*1011 / 6.02*1023 plasmid moles in 1 ml: = 10 -12 mol plasmid in 1 ml
Each plasmid molecule has a molecular weight of 660 g/mol x 8000 bp = 5.28 *106 g/mol
for one plasmid
In 1 ml, we have 10-13 mol plasmid * 5.28 *106 g/mol = 5.28 *10-6 g plasmid (=5.28µg)
About 5 µg will be isolated from one ml, if you want 10 µg and calculate 50% loss, you
need at least 4 ml.

6. After performing plasmid DNA according to the appropriate protocol, you measured a DNA
concentration of 200ng/mL for mCherry and 250ng/mL for tdTomato. In both samples, you
measure an absorption ratio at A260nm/A280nm of 1.75.
Have you isolated pure DNA or are there contaminations with other biomolecules in your
solution? If so, with what? Explain your answer.
Answer:
As the A260nm/A280nm is a bit lower than 1.8, the sample is a bit contaminated with proteins.

PCR 16 point (2,1,1,4,2,3,2,1)

The lab works with the plasmid p-OFP, this plasmid contains the gene that encodes an Orange
Fluorescent Protein. Experience shows that mutations can occur on 3' side of the gene. That is why it
is important that the plasmid DNA that you have isolated is checked using the PCR method. For this
experiment you have chosen primers that bind at the beginning and end of the gene, when a
mutation has occurred, 1 of the primers cannot bind.

p-OFP
6842 bp

OFP-gene
1. For this question, use the codon table below.
When the mutation to the OFP gene is created, the amino acid Tryptophan is replaced by
Glycine in the OFP protein. Give the code on the triplet encoding tryptophan and show which
nucleotide needs to be changed so that it encodes glycine.
Triplet coderend for tryptophan: TGG
The T needs to be changed into a G, then you will get GGG coding for glycine.

2. How do you call the type of mutation described in question 1

Point mutation

3. Due to this mutation, the Orange Fluorescent Protein no longer fluoresces. Explain how this is
possible.
Due to the mutation the folding of the protein (secundair and tertiair) structure can change. A
misfolded protein can lose its function, in this case fluoresce.
4. To perform the PCR reaction on the OFP gene you need to make a pipetting scheme for 1
reaction.
According to your protocol, a PCR reaction contains the following reagents in the following
concentrations/quantities:
o 1x PCR reaction buffer
o 1,5 mM MgCl2 solution
o 0,2 mM dNTP solution,
o 0,5 µM forward primer
o 0,5 µM reverse primer
o 0,5U Taq DNA polymerase
o 5 µL of DNA (different concentrations)
o Add Milli-Q water to a final volume of 25 µL

In the freezer you can find the following reagents:

o 5x PCR reaction buffer
o 25 mM MgCl2 solution
o 1,25 mM dNTP solution,
o 10 µM forward primer
o 10 µM reverse primer
o 5U/µL Taq DNA polymerase
You use 5 µL of the isolated plasmid DNA.

a) Make a pipetting scheme for one sample?

Answer:
o 5 µL PCR reaction buffer
o 1,5 µL MgCl2 solution
o 4 µL dNTP solution,
o 1,25 µL forward primer
o 1,25 µL reverse primer
o 0,1 µL Taq DNA polymerase
o 6,9 µL Water

b) Explain which controls you will use in the PCR experiment.

Answer:
A negative control in which water is added instead of DNA to check whether the mix is
contaminated.
A positive control with a DNA sample that will definitely work in the PCR reaction (p-OFP
without mutation)

5. For the PCR you want to use primers available in the lab. You first check with the help of a
program such as CLC-bio whether these are good primers.
a) What are 3 characteristics of good primers?
CG content 40-60%, length 18-25bp, CG clamp, ∆Tm maximal 5C
 b) Describe at least 1 part of your experiment preparation where you used bioinformatics.
In CLC you can do 3 things: Analyze primer properties in CLC, run the PCR in CLC and thus
determine the expected fragment size, calculate Tm value of the primers to calculate the
annealing temperature
But database use is also a good answer here: looking up the sequence of the plasmid in
NCBI/ Genbank (and then putting it in CLC etc).

6. For this PCR reaction you have these 2 primers:

Forw: 5’- ATATG CACAT GAAGC TGTAC - 3', ,binds to positions 1294-1314bp
Rev: 5’ - GCTAT CAGTC AGTCA GTCG -3’, binds to positions 1791-1810bp
a) What is the expected size of the PCR product?
1810-1294+1 =517 bp

Chromatography 9 points (2, 2, 3, 2)

We have the following two peptides:

Peptide 1: Amino terminal - Lys- Ala- Ser- Arg- Trp- Carboxyl terminal

Peptide 2: Amino-terminal - Cys- Ala- Glu- Ala- Lys - Asp - Pro-Carboxyl terminal

a. Peptide 1 contains the amino acid Lysine. Draw the Lewis structure of Lysine at pH4.
 b. Determine the net charge of these two peptides at pH 6. Show the partial charges of
the amino acid residues and the total charge of the peptide.
Peptide 1 at pH 6:

Amino- Lys Ala Ser Arg Trp Carboxyl- TOTAL

terminal terminal
+1 +1 0 0 +1 0 -1 2+

Peptide 2 at pH 6:

Amino- Cys Ala Glu Ala Lys Asp Pro Carboxyl- TOTAL
terminal terminal
+1 0 0 -1 0 +1 -1 0 -1 -1

c. We want to separate a mixture of these two peptide by using ion exchange

chromatography
Describe how you will do that. In the description should be included: the type of
column, (anion or cation) the starting buffer pH, the charges of the peptides at this
pH, the elution buffer and the order of elution.

Example of a correct answer:

Take an anion column and a starting buffer at pH 6. At that pH peptide 1 is positively
charged while peptide 2 is negatively charged. Therefore, peptide 1 will bind to the
column while peptide 2 will elute. Increase afterwards the salt concentration of the
buffer. Peptide 1 will elute too.

d. Peptides 1 and 2 could also be separated by size exclusion chromatography (SEC).

Peptide Molecular
weight
1 1000
2 1500
Explain which column range would you use to separate the two peptides with SEC.
What is the elution order?

Example of a correct answer:

Pore size :1000. Peptide 1 can into the pores and therefore will take longer to be
eluted from the column compared to peptide 2.
Elution order: peptide 2 / Peptide 1
Buffers 10 points (1,3,2,2,2)
During the lab class you have to prepare a SDS-Poly-Acrylamide-Gel (SDS-PAGE). This gel
contains, among others, a Tris-HCl buffer with a pH of 8.8. There is no more buffer left in the
lab so you have to prepare a new one. To do that you dissolve 1.5 mol Tris-HCl in water and
then you titrate by using a 5.00 M NaOH solution until you reach the desired pH. After that,
you add water to make 1 L of the buffer solution. The pKa of Tris-HCl is 8.1

a) Within what pH range can you use a Tris-HCl buffer?

7.1-9.1
b) Calculate the ratio of Tris-HCl and it’s conjugated base that have to be present in this
buffer.
pH = pKa + log [A-]/[HA].
8.8= 8.1 + log [A-]/[HA].
0.7 = log [A-]/[HA]
5.01= [A-]/[HA]

c) Draw the Lewis structures of CF4 and CHF3.

d) Explain if there will be a difference in solubility in water between CF4 and CHF3.
CF4 is apolar and CHF3 is polar/is a dipole molecule, associated conclusion that CHF3 is
therefore a polar molecule/dipolar molecule and can therefore form hydrogen bonds
with water so better dissolves in water compared to CF4.

e) At the protein structure level, explain why GFP no longer fluoresces when the pH is
below 4.5.
GFP has a primary, secondary and tertiary structure.
At high or low pH, the charges of the side groups of amino acids can change. This
changes tertiary bonds such as ionic bonds and hydrogen bonds. The tertiary structure
will change as a result/the protein may denature/is no longer properly folded so its
function, in this case fluorescence, is lost.
Analysis of proteins 9 points (2, 2, 2, 1, 2)
After isolation and purification experiments you want to determine the amount of PFP protein. For
this you use a number of experiments: the nanodrop, a concentration determination and SDS-poly-
acrylamide gel electrophoresis.

You were planning to use the Bradford method for the concentration determination. Unfortunately,
the reagents for the Bradford protein determination have run out. Your colleague suggests using the
Lowry; Because the reagents are in stock for this.

a. What is the advantage and disadvantage of the Lowry method compared to the Bradford
method?
Advantage: measures a large spectrum of protein concentrations
Disadvantage: can be disturbed by organic buffers or ammonium sulphate

b. What should you do so you can still use the Lowry properly, despite these disadvantages?
Ensure that the protein is not in an organic buffer or ammonium sulphate, for example by
precipitation of the protein, dilution or dialysis.

Using SDS-PAGE proteins are separated based on size. The 3D structure of a protein also affects the
speed at which it migrates through the gel and therefore the 3D structure of the protein is broken
before the sample is put on gel. This is done by adding sample buffer and boiling the sample.

c. Explain which protein structure(s) are broken by the sample buffer and boiling up the
sample?.
Primary structure – Secondary structure – Tertiary structure
circle the correct answer(s))
Explanation (indicate in your answer the bonds that are broken):

Both the secondary and tertiary structures are broken. During denaturation, all non-covalent
interactions (such as hydrogen bonds, vanderwaals interactions, ion-ion interactions, dipole-
dipole interactions) are broken. The covalent peptide bonds (part of primary structure)
remain intact.

However in the cells it is important that the proteins are folded properly.

d. What is the name of the proteins that help in the process of folding proteins?
Chapero(e) proteins
e. What happens in the cell with a improperly folded protein?
Possible answers:
• These proteins are ubiquitinated.
• (these proteins are ubiquitinated), then broken down by the proteasome.
• These proteins are refolded into special chaperone proteins
Protein properties 6 points (2,2,2)
During molecular biology lessons you learned that transcription factors bind to DNA.

a. Which type of amino acids (which properties must their rest groups have) must be present in
the transcription factors to realize this? Explain your answer.
DNA is negatively charged, thus transcription factors should be positively charged and
contain thus mainly basic amino acids

b. Do you expect the GFP part of the protein to be attracted to the DNA or do you expect it to
be as far away as possible from the DNA? Explain your answer based on the properties of
GFP and the properties of the DNA.
GFP is a hydrophobic protein and this will be far away from hydrophilic molecules, such as
the negatively charged DNA, thus it will be as far away from the DNA as possible

c. Do you expect the GFP to be fluorescent at the pH that is present in the cells (around 7.4)?
Explain your answer.
Yes, because GFP is fluorescent at an pH of 6,2 or higher.
 Appendix Biochemistry

Periodic Table
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

1 2
1s 1 H
1.008
He
4.003
3 4 5 6 7 8 9 10
2s, 2p 2 Li
6.941
Be
9.012
B
10.81
C
12.01
N
14.01
O
16.00
F
19.00
Ne
20.18
11 12 13 14 15 16 17 18
3s, 3p 3 Na
22.99
Mg
24.31
Al
26.98
Si
28.09
P
30.97
S
32.07
Cl
35.45
Ar
39.95
19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
4s, 3d, 4p 4 K
39.10
Ca
40.08
Sc
44.96
Ti
47.88
V
50.94
Cr
52.00
Mn
54.94
Fe
55.85
Co
58.93
Ni
58.69
Cu
63.55
Zn
65.39
Ga
69.72
Ge
72.61
As
74.92
Se
78.96
Br
79.90
Kr
83.80
37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54
5s, 4d, 5p 5 Rb Sr Y Zr Nb Mo Tc Ru Rh Pd Ag Cd In Sn Sb Te I Xe
85.47 87.62 88.91 91.22 92.91 95.54 (99) 101.1 102.9 106.4 107.9 112.4 114.8 118.7 121.8 127.6 126.9 131.3
55 56 57* 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86
6s, 4f, 5d, 6p 6 Cs Ba La Hf Ta W Re Os Ir Pt Au Hg Tl Pb Bi Po At Rn
132.9 137.3 138.9 178.5 180.9 183.9 186.2 190.2 192.2 195.1 197.0 200.6 204.4 207.2 209.0 (209) (210) (222)
87 88 89** 104 105 106 107 108 109 110 111 112 114 116 118
7 Fr Ra Ac Rf Db Sg Bh Hs Mt Uun Uuu Uub Uuq Uuh Uuo
(223) 226.0 227.0 (261) (262) (263) (262) (265) (268) (269) (272) (277) (289) ?? (289) (293)

58 59 60 61 62 63 64 65 66 67 68 69 70 71
* Lanthaniden
Ce Pr Nd Pm Sm Eu Gd Tb Dy Ho Er Tm Yb Lu
140.1 140.9 144.2 (145) 150.4 152.0 157.3 158.9 162.5 164.9 167.3 168.9 173.0 175.0
90 91 92 93 94 95 96 97 98 99 100 101 102 103
** Actiniden
Th Pa U Np Pu Am Cm Bk Cf Es Fm Md No Lr
232.0 231.0 238.0 237.0 (244) (243) (247) (247) (251) (252) (257) (258) (259) (260)
 pKa α-carboxyl pKa α-amino pKa-side Structure
group group chain

Gly Glycine 2.34 9.60

Ala Alanine 2.35 9.87

Ile Isoleucine 2.32 9.76

Leu Leucine 2.33 9.74

Val Valine 2.29 9.74

Phe Phenylalanine 1.83 9.13

Trp Tryptophan 2.38 9.39

Pro Proline 1.99 10.96

Tyr Tyrosine 2.41 8.67 11.01 O

OH
NH2
HO
Cys Cysteine 1.7 10.47 8.36 O

HS OH
NH2

Met Methionine 2.13 9.28

Ser Serine 2.21 9.15

Thr Threonine 2.09 9.10

Asn Asparagine 2.14 8.72

Gln Glutamine 2.19 9.00

Asp Aspartic acid 2.10 9.82 3.86 O

HO
OH
O NH2
Glu Glutamic acid 2.16 9.96 4.3 O O

HO OH
NH2
Arg Arginine 1.823 8.991 12.1 NH O

H 2N N OH
H
NH2
His Histidine 1.77 9.18 6.10 O
N
OH
HN NH2

Lys Lysine 2.18 8.95 10.33 O

H 2N
OH
NH2
Table of acids and bases

In water, T= 298 K

Acid Ka pKa Base Kb pKb

HClO4 >>1 <0 ClO4- <<10 -14 >14
HI >>1 <0 I- <<10 -14 >14
HBr >>1 <0 Br- <<10 -14 >14
HCl >>1 <0 Cl- <<10 -14 >14
H2SO4 >>1 <0 HSO4- <<10 -14 >14
HNO3 >>1 <0 NO3- <<10 -14 >14
HClO3 >>1 <0 ClO3- <<10 -14 >14
H3O+ H2O
CCl3-COOH 2.2 ·10-1 0.66 CCl3-COO- 4.6 ·10-14 13.34
H2CrO4 1.8 ·10-1 0.74 HCrO4- 5.5 ·10-14 13.26
HIO3 1.7 ·10-1 0.78 IO3- 6.0 ·10-14 13.22
H2C2O4 5.6 ·10-2 1.25 HC2O4- 1.8 ·10-13 12.75
CHCl2-COOH 4.4 ·10-2 1.35 CHCl2-COO- 2.2 ·10-13 12.65
H3PO3 1.6 ·10-2 1.80 H2PO3- 6.3 ·10-13 12.20
SO2 + H2O (H2SO3) 1.4 ·10-2 1.85 HSO3- 7.1 ·10-13 12.15
HClO2 1.1 ·10-2 1.94 ClO2- 8.7 ·10-13 12.06
HSO4- 1.0 ·10-2 1.98 SO42- 9.5 ·10-13 12.02
H3PO4 6.9 ·10-3 2.16 H2PO4- 1.4 ·10-12 11.84
H3AsO4 5.5 ·10-3 2.26 H2AsO4- 1.8 ·10-12 11.74
NH3+ -CH2 – COOH 4.5 ·10-3 2.35 NH3+ -CH2 – COO- 2.2 ·10-12 11.65
CH3-CHCl-COOH 2.3 ·10-3 2.83 CH3-CHCl-COO- 6.8 ·10-12 11.17
Fe(H2O)6 3+ 1.5 ·10-3 2.83 FeOH(H2O)5 2+ 6.8 ·10-12 11.17
HOOCCH2COOH 1.5 ·10-3 2.85 HOOCCH2COO- 7.1 ·10-12 11.15
CH2Cl-COOH 1.4 ·10-3 2.87 CH2Cl-COO- 7.4 ·10-12 11.13
H3C6H5O7 7.4 ·10-4 3.13 H2C6H5O7- (H2C-) 1.3 ·10-11 10.87
(citric acid)
HF 6.3 ·10-4 3.20 F- 1.6 ·10-11 10.80
HNO2 5.6 ·10-4 3.25 NO2- 1.8 ·10-11 10.75
HCOOH 1.8 ·10-4 3.75 HCOO- 5.6 ·10-11 10.25
HC2O4- 1.6 ·10-4 3.81 C2O42- 6.3 ·10-11 10.19
Cr(H2O)6 3+ 1.5 ·10-4 3.82 CrOH(H2O)5 2+ 6.6 ·10-11 10.18
CH3CHOHCOOH 1.4 ·10-4 3.86 CH3CHOHCOO- 7.2 ·10-11 10.14
H2Se 1.3 ·10-4 3.89 HSe- 7.8 ·10-11 10.11
CH2Cl-CH2-COOH 1.0 ·10-4 3.98 CH2Cl-CH2-COO- 9.5 ·10-11 10.02
H2C6H6O6 (ascorbic 9.1 ·10-5 4.04 HC6H6O6 - (HA-) 1.1 ·10-10 9.96
acid = vitamin C)
C6H5COOH 6.3 ·10-5 4.20 C6H5COO- 1.6 ·10-10 9.80
CH3-COOH 1.7 ·10-5 4.76 CH3-COO- 5.8 ·10-10 9.24
H2C6H5O7- (H2C-) 1.7 ·10-5 4.76 HC6H5O72- (HC2-) 5.8 ·10-10 9.24
CH3CH2CH2COOH 1.5 ·10-5 4.83 CH3CH2CH2COO- 6.17 ·10-10 9.17
 Acid Ka pKa Base Kb pKb
CH3-CH2-COOH 1.4 ·10-5 4.87 CH3-CH2-COO- 7.4 ·10-10 9.13
C6H5-NH3+ 1.4 ·10-5 4.87 C6H5-NH2 7.4 ·10-10 9.13
Al(H2O)6 3+ 9.8 ·10-6 5.01 AlOH(H2O)5 2+ 1.0 ·10-9 8.99
HOOCCH2COO- 2.0 ·10-6 5.70 -OOCCH COO-
2 5.0 ·10-9 8.30
CO2 + H2O (H2CO3) 4.5 ·10-7 6.35 HCO3- 2.2 ·10-8 7.65
HC6H5O72- (HC2-) 4.0 ·10-7 6.40 C6H5O72- (C3-) 2.5 ·10-8 7.60
HCrO4- 3.2 ·10-7 6.49 CrO42- 3.1 ·10-8 7.51
Fe(H2O)6 2+ 1.8 ·10-7 6.74 FeOH(H2O)5 + 5.5 ·10-8 7.26
H2AsO4- 1.7 ·10-7 6.76 HASO4 2- 5.8 ·10-8 7.24
H2S 8.9 ·10-8 7.05 HS- 1.1 ·10-7 6.95
H2PO4- 6.2 ·10-8 7.21 HPO4 2- 1.6 ·10-7 6.79
HSO3- 6.2 ·10-8 7.21 SO32- 1.6 ·10-7 6.79
HClO 4.0 ·10-8 7.40 ClO- 2.5 ·10-7 6.60
Cu(H2O)6 2+ 1.0 ·10-8 8.00 CuOH(H2O)5 + 1.0 ·10-6 6.00
HBrO 2.8 ·10-9 8.55 BrO- 3.5 ·10-6 5.45
Zn(H2O)6 2+ 1.1 ·10-9 8.96 ZnOH(H2O)5 + 9.1 ·10-6 5.04
HCN 6.1 ·10-10 9.21 CN- 1.6 ·10-5 4.79
H3AsO3 6.0 ·10-10 9.22 H2AsO3- 1.7 ·10-5 4.78
NH4+ 5.6 ·10-10 9.25 NH3 1.8 ·10-5 4.75
H3BO3 5.4 ·10-10 9.27 H2BO3- 1.9 ·10-5 4.73
NH3+-CH2-COO- 1.7 ·10-10 9.78 NH2-CH2-COO- 6.0 ·10-5 4.22
(CH3)3NH+ 1.6 ·10-10 9.80 (CH3)3N 6.3 ·10-5 4.20
C6H5OH 1.0 ·10-10 9.99 C6H5O- 9.8 ·10-5 4.01
HCO3- 4.7 ·10-11 10.33 CO3 2- 2.1 ·10-4 3.67
HIO 2.3 ·10-11 10.64 IO- 4.4 ·10-4 3.36
C2H5NH3+ 2.3 ·10-11 10.65 C2H5NH2 4.5 ·10-4 3.35
CH3-NH3+ 2.2 ·10-11 10.66 CH3-NH2 4.6 ·10-4 3.34
(CH3)2NH2+ 2.2 ·10-11 10.73 (CH3)2NH 5.4 ·10-4 3.27
HAsO4 2- 1.9 ·10-11 11.29 AsO4 3- 1.9 ·10-3 2.71
H2O2 5.1 ·10-12 11.62 HO2- 4.2 ·10-3 2.38
HC6H6O6 - (HA-) 2.4 ·10-12 11.75 C6H6O6 - (A2-) 5.6 ·10-3 2.25
HPO4 2- 1.8 ·10-12 12.32 PO4 3- 2.1 ·10-2 1.68
H2O 4.8 ·10-13 OH-
HS- <<10-14 >14 S2- >>1 <0
CH3-CH2OH <<10-14 >14 CH3-CH2O- >>1 <0
H2 <<10-14 >14 H- >>1 <0
NH3 <<10-14 >14 NH2- >>1 <0
OH- <<10-14 >14 O2- >>1 <0

Henderson-Hasselbalch equation: pH = pKa + log [A-]/[HA]

Appendix II

Protocol A

Materials
• Disposables: sterile vials (1.5 mL) and sterile pipette tips
• Micro pipettes
• Micro centrifuges
• Centrifuge and buckets
• Speed vac: apparatus to dry DNA samples
• Heating block or oven at 37 oC
Solutions
LB medium: 10 g bacto tryptone, 5 g bacto yeast extract, 10 g NaCl. Dissolve in 900 mL
MiliQ, adjust pH to 7.0 and the volume to 1000 ml. Autoclave for 20 minutes
at 121 oC
THMS solution: 30 mM Tris-HCl (pH 8.0), 3 mM MgCl2, 25 % sucrose.
Lysozyme solution: 2 mg/mL lysozyme in THMS.
Proteinase K: 20 mg/mL proteinase K in sterile MQ water, stored at - 20 °C.
TES solution: 50 mM Tris-HCl (pH 8.0), 5 mM EDTA, 50 mM NaCl.
TES - SDS: dissolve 50 mg SDS (sodium dodecyl (lauryl) sulphate) in 10 mL TES.
Tris solution: 10 mM Tris-HCl pH 7.5, 15 mM NaCl.
RNase A: 10 mg/mL in 50 mM Tris/HCl (pH7.0). The solution is heated for 15 minutes
at 100 °C and cooled slowly to room temperature. Aliquots of 1 mL are
stored -20 °C.
Chloroform : Isoamyl alcohol: 24 : 1
3 M NaAc: 3 M sodium acetate (pH5.6) in miliQ.
96 % ethanol
70 % ethanol
TE buffer: 10 mM Tris-HCl (pH 8.0), 1 mM EDTA; sterilized by autoclaving.

Procedure

• Inoculate 50 mL LB medium with a few fresh colonies from a plate and grow to obtain sufficient
density.
For E. coli: LB medium at 37 oC, 250 rpm, overnight.
• Pellet cells of an overnight culture in a suitable centrifuge tube/bucket by centrifugation for 10
minutes at 4,000 rpm.
• Remove the supernatant (bacterial waste container) and resuspended the cell pellet in 10 mL
THMS.
• Pellet the cells again by centrifugation for 10 minutes at 4000 rpm.
• Remove the supernatant (bacterial waste container) and resuspended the cell pellet in 10 mL
THMS with lysozyme. Incubate for minimal 45 minutes at 37 oC
• Centrifuge for 5 minutes at 6,000 rpm.
• Carefully remove the supernatant without disturbing the pellet (bacterial waste container).
• Resuspend the pellet in 1000 uL THMS and aliquot into 100 uL portions in 1.5 mL vials (sterile).
• Add to each vial 10 uL RNase solution, mix and add 390 uL TES with 0.5 % SDS. A clear solution
with a “white cloud” will be visible.
• Add 50 uL proteinase K and incubate for minimally 1 hr (preferably overnight) at 37 oC.
• Add 500 uL chloroform-isoamylalcohol and mix carefully but thoroughly.
• Centrifuge for 5 minutes at 13,000 rpm.
• Transfer 450 µL of the upper layer without the interphase to a new 1.5 mL vial. Use a yellow tip
of which you cut the end.
• Repeat the extraction with chloroform : isoamylalcohol twice, transferring 375 µL the second
time and 300 µL the third time.
• Add 20 uL 3M NaAc solution and 750 µL 96% ethanol to each tube and mix carefully but
thoroughly.
• After 5 minutes at room temperature centrifuge for 10 minutes at 13.000 rpm.
• Carefully remove the supernatant (using vacuum pump) without disturbing the DNA pellet.
• Add 500 uL 70% Ethanol to each vial, mix and centrifuge for 1 minute.
• Remove all of the supernatant without disturbing the DNA pellet. Spin again if needed.
• Dry the DNA pellets in the speed vac or in a 37 °C oven.
• Pipette 100 uL TE solution per vial and store the vials overnight in the cold room (+ 4 oC) to
dissolve the chromosomal DNA.
• Combine the solutes and store at - 20 oC.
• The DNA content and purity can be measured using protocol 'Determination of DNA
concentration and purity'.
• For determination of the integrity (average size) of the DNA, run an aliquot on a 0.7 % agarose
gel with high-range molecular size markers.
Protocol B

Materials
• Overnight culture of E. coli containing the right plasmid in LB with antibiotic
• Microcentrifuge/Eppendorf vials (1.5 and 2.0 mL)
• Clean and sterile centrifuge tubes
• Micro-pipettes and sterile tips
• Ice
• Eppendorf centrifuge
• Water bath 37 oC
Solutions
Washing solution NTE 10 mM Tris/HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA.
RNase A 10 mg/mL in 10 mM Tris/HCl (pH 7.5), 15 mM NaCl.
Solution I: TEG 25 mM Tris/HCl (pH 8.0), 10 mM EDTA, 50 mM glucose, 100 µg/mL RNase
A.
Solution II 0.2 M NaOH, 1% SDS. Prepare freshly from 2 M NaOH and 10 % SDS
solutions.
5 M KAc 3 M Potassium acetate, 2 M acetic acid.
Chloroform/Isoamyl alcohol 24/1 (v/v) saturated with 0.1 M Tris/HCl (pH 8.0)
3 NaAc 3 M sodium acetate (pH 5.6). Dissolve in miliQ and adjust with 100 % acetic
acid.
TE buffer 10 mM Tris/HCl (pH 8.0), 1 mM EDTA.
TE-RNase 20 µg/mL RNase A in TE buffer.

Procedure
• Retrieve the overnight culture from the incubator. Transfer 1.5 mL of the culture into 2 micro-
centrifuge tubes of 2.0 mL and centrifuge for 1 minute at 13,000 rpm.
• Remove the supernatant with a micro pipette. This is collected is a bacterial waste container.
• Repeat previous steps two times for the same plasmid in the same micro-centrifuge tubes.
• Each micro-centrifuge tube contains now a bacterial pellet from 4.5 mL culture.
Show the practical instructor the size of the bacterial pellet. If this is too small repeat first two
steps until the size of the pellet is sufficient!
(a small pellet means less bacterial cells and as a consequence less plasmid DNA!)
• Store the remainder of the culture at 4 °C as back-up.
• Wash the bacteria by re-suspending them in 0.5 mL ice-cold NTE buffer, centrifuging for 1 min. at
13,000 rpm and removing the supernatant as described before.
• Re-suspend the pellet in 400 µL solution I (with RNase).
• Add 400 µL solution II (freshly made). Homogenise the mixture by inverting the tubes and
incubate for max. 5 minutes at room temperature. The mixture should immediately become
transparent and viscous.
• Add 400 µL ice-cold potassium acetate (KAc) pH 4.8 to neutralize the mixture. The content of the
vial is homogenized again by inverting the tubes and incubated for about 5 minutes on ice (a
white precipitate is formed).
• Centrifuge for 5 minutes at 13,000 rpm and transfer the supernatant (cleared lysate) into a
sterile 2.0 mL micro-centrifuge tube. DO NOT TRANSFER ANY OF THE PRECIPITATE !
• Add 0.6 volume isopropanol, mix by inverting and leave for 2 - 5 minutes at room temperature.
• Centrifuge for at least 15 minutes at 13,000 rpm. Remove the supernatant (by means of a small
tip and a vacuum pump).
• Add 1.0 mL 70% ethanol to the DNA pellet, shake for a few seconds and centrifuge for 2 minutes
at room temperature.
• Remove carefully the supernatant and dry the pellet under vacuum. The smell of ethanol should
have disappeared.
• Dissolve the pellets in 100 µL TE buffer pH 8.0 + RNase (20 µg/mL), combine in one 1.5 mL tube
and incubate at 37 °C for 15 minutes.
• Add an equal volume of chloroform/isoamylalcohol, shake vigorously and spin for 2 minutes to
separate the phases.
• Transfer the water phase to a clean tube. Avoid any material from the interphase! Add 10 µL 3M
sodium acetate (NaAc) and 500 µL absolute ethanol. Incubate at -20 °C (-80 °C is even better) for
at least 1 hour, but preferably overnight.
• Centrifuge for at least 15 minutes at 13,000 rpm. Remove the supernatant using a capillary.
• Wash the pellet with 1.0 mL 70% ethanol and centrifuge for 1 minute at room temperature.
• Remove the supernatant and dry the pellet under vacuum.
• Add 50 µL TE buffer pH 8.0 to the pellet and dissolve plasmid DNA.
• Code the vials with the name of the sample, date and student name.
• Store coded DNA solutions in a freezer.
• To determine the DNA concentration; see protocol Determination of genomic DNA concentration
and purity.
• To determine if you isolated of the right plasmid DNA, digest 1 µL of plasmid DNA solution with a
cheap restriction enzyme that cuts the plasmid DNA only once. By means of Agarose gel
electrophoresis you can obtain qualitative as well as quantitative information; see Agarose gel
electrophoresis.