1. RESEARCH SCHOOL OF CHEMISTRY
THE AUSTRALIAN NATIONAL UNIVERSITY
LABORATORY MANUAL CHEM3204
PRACTICAL COURSE 2016
Making and analysing tRNA synthetases in vivo and
cell-free
1

2. Timetable
Mutagenesis & Transformation . Plasmid prep & Sequencing . Expression
Monday Tuesday Wednesday Thursday Friday
Week
1
9 am TH
introduction
1. Point
mutagenesis
PCR of RS (3 h)
10 am GO
lecture/PCR
tute
1 pm 2. PCR
purification and
concentration
measurement
(1 h)
3. Agarose gel
electrophoresis
4. RQ assembly
and
transformation
(1.5 h)
4 pm TH course
aims
9 am
Documenting the
results and start
of overnight cell
culture (0.5 h)
10 am TH
lecture (2 h)
1 pm GO PBL/
TH MD prac
9 am
1. Plasmid
miniprep (0.5
h)
2. DNA
concentration
determination
(1 h)
3. Sequencing
PCR (3.5 h)
11 am GO
lecture (1h)
2 pm
4. Reaction
clean up and
submission to
BRF
9 am
TH lecture (2 h)
11 am
GO primer
software
1. Sequence
alignment (1 h)
2. Transformation
of XJB(DE3) cells
(0.5-1 h)
9 am
Inoculation
of start
culture
(0.5 h)
9:30
GO lecture (2
h)
1:30 pm GO
PBL/ TH MD
prac
Expression Purification . Cell-free . Analysis
Week
2
9 am
1. Cell culture
inoculation (0.5
h)
2. Cell growth
to OD600 0.7-1.0
and sampling
(3 h)
10 am GO
protein purif.
lecture
9 am
1. Affinity
chromatography
(2 h)
2. SDS-PAGE,
staining and
destaining (2 h)
3. Protein
concentration
determination
(1 h)
9 am TH
Molecular
dynamics
Lecture/tute
(3 h)
2 pm
Cell-free
protein
synthesis,
reaction set up
(2 h)
9 am
SDS-PAGE
electrophoresis
and staining,
destaining (2h)
11 am
TH Lecture/tute
(4 h)
9 am
PBL
presentation
s (3 h)
1 pm course
summary /
rapup
2

3. 3. IPTG
induction (3.5
h)
PBL study time
4. Sampling and
cell pelleting
(0.5 h)
GO tute primers
for cell-free
Tute UAA
modelling with
ChemDraw and
PyMOL
GO lecture
cryoelectron
microscopy
Table of Contents
INTRODUCTION 6
Safety and Laboratory Rules 9
EXPERIMENTAL PROTOCOLS 13
Monday 13
Site-directed mutagenesis 13
Point mutagenesis of aminoacyl-tRNA synthetase gene by PCR 13
Agarose gel electrophoresis - Measuring DNA concentration 14
Purification of the PCR product 14
RQ vector assembly 15
E. coli transformation 15
Tuesday 15
Count the transformants 15
Overnight cell culture 16
3

4. Wednesday 16
Plasmid preparation and sequencing 16
Plasmid miniprep and DNA concentration determination 16
Sequencing PCR 18
PCR purification and submission for sequencing 18
Thursday 18
Confirmation of the mutant DNA sequence 18
Transformation 21
Friday 20
Protein overexpression in vivo 20
Inoculation of start culture 20
Monday 20
Inoculation of cell culture 20
Cell growth to OD600 0.7-1.0 20
Induction with IPTG 21
Sampling and cell pelleting 22
Tuesday 22
Protein purification 22
Ni-NTA affinity chromatography 22
SDS-PAGE 23
Protein concentration determination 23
4

5. Wednesday 23
Cell-free protein synthesis 23
Setup of cell-free reaction 24
Thursday 25
Analysis of the cell-free protein expression yield 25
SDS-PAGE 25
Appendix A: Working under sterile conditions 27
Appendix B: NEB 2-log DNA ladder 28
Appendix C: DNA concentration determination by NanoVue 29
Appendix D: Protein purification using a His GraviTrap column 30
Appendix E: SDS-PAGE 31
Appendix F: BioRad protein molecular weight markers 33
Appendix G: Making amino acid mixtures 34
Appendix Chemical Risk Assessment 35
5

6. INTRODUCTION
This intensive course is directed at students who intend to make and use proteins. It is
planned as a stand-alone course that, in the future, may include non-ANU students
too.
Two approaches will be practiced:
1. In vivo protein expression
2. Cell-free protein synthesis
In addition, the practicals include site-directed mutagenesis.
About in vivo protein expression
This is the traditional way of making proteins. E. coli cells are transformed with an
overexpression vector, a large number of cells are grown, production of the target
protein is induced with IPTG, and after a few hours of expression the cells are
harvested and the protein extracted from the cells.
To allow induction with IPTG, the E. coli genome contains the gene for the T7 RNA
polymerase under control of the lac operon. The gene of the target protein is on a T7
vector, i.e. will be transcribed by T7 RNA polymerase but not by the E. coli RNA
polymerase. The T7 RNA polymerase generates a large amount of mRNA of the
target gene and the target protein can make up to 50% of the total protein of the cells.
To isolate the protein, the cells must be lysed and the protein purified. In this practical
we make the target protein with a His6-tag for immobilized metal affinity
chromatography (IMAC).
Analysis of protein yield and purity is by NanoVue measurement and SDS-PAGE
electrophoresis.
About cell-free protein synthesis
Cell-free protein synthesis (CFPS) yields less protein but is much faster than in vivo
expression. Our CFPS system uses an E. coli cell extract, which is prepared by
growing a lot of E. coli in a fermenter, turning the cells inside-out by pressing them
through a small pore (French Press) and centrifuging at high speed to spin down the
cell-wall debris. This results in the so-called S30 extract. You will be provided with
S30 extract.
S30 extracts contain all the necessary enzymes for coupled transcription/translation,
i.e. the target gene template DNA provided is transcribed into mRNA and the mRNA
is translated into the target protein. The extract contains T7 RNA polymerase.
Suitable DNA constructs contain a T7 promoter and a T7 terminator, a Shine-
Dalgarno sequence for ribosome binding, and a target gene beginning with an ATG
start codon and ending with a TAA or TGA stop codon. Conventional expression
vectors work perfectly fine, but linear PCR products can be used too.
(Comment: S30 extracts contain all sorts of nucleases. To avoid digestion of linear
PCR products, mainly by exonucleases, these need to be made with matching
phosphorylated 8-base overhangs so that the PCR product can be cyclised by the
6

7. ligase that is also present in the S30 extract. In this course, however, the cell-free
reaction will use plasmid DNA.)
Cell-free protein synthesis can produce milligram-per-mL protein yields. This amount
is sufficient for analysis by nuclear magnetic resonance (NMR) spectroscopy on
modern high-field NMR spectrometers, providing a convenient and rapid way to
analyze proteins.
In this course, the reaction product will be analysed by SDS-PAGE.
Site-directed mutagenesis
Site-directed mutagenesis will use an improved Quikchange protocol, which we refer
to as RQ (for reliable and quick). The method uses two mutation primers to amplify
the vector by PCR, an exonuclease to generate single-stranded DNA overhangs, and
the capacity of E. coli to fill in missing nucleotides and ligate the vector in vivo.
Our ambition
Each student gets to produce a different mutant aminoacyl-tRNA synthetase,
abbreviated RS. The RS enzymes were computationally designed to charge
suppressor tRNA with phosphotyrosine (something that has never been achieved
before). The suppressor tRNA recognises the amber stop codon. The cell-free protein
synthesis experiment at the end of the second week will use your purified RS together
with suppressor tRNA to try and incorporate a phosphotyrosine analogue into a target
protein, the gene of which was engineered to contain an amber stop codon.
7

8. If this works, it will be a publication! (In other words, don’t be disappointed if the
final cell-free reaction produces no full-length protein.)
What is in it for you
The wet-lab programme is designed to train you in site-directed mutagenesis, in vivo
protein synthesis and cell-free protein synthesis in an intensive course. This is a lot of
ground to be covered – even experienced researchers can take 2 weeks to make a
protein mutant by in vivo protein expression.
Spare time between experiments will be filled with lectures, tutes and report writing.
New skills
Working under sterile conditions
Working with E. coli cell cultures
In vivo protein expression
Using cell-free protein synthesis
Site-directed mutagenesis
Agarose gel electrophoresis
DNA sequencing
Refresh from CHEM2208
Plasmid preparation
Transformation
SDS-PAGE
Concentration determination
8

9. SAFETY
Chemical hazards
A risk assessment has to be performed before each experiment via Chemwatch (see
below).
Biological hazards
Even though these practicals, like the overwhelming majority of research projects at
the RSC, use exclusively low-risk laboratory strains of E. coli that have been used for
decades, these bacterial cells are live microorganisms. Whenever microorganisms are
used, it is essential that laboratory workers strictly adhere to a microbiology
laboratory code of practice and thereby significantly reduce the possibility of causing
a laboratory-acquired infection.
Note that bacterial cell cultures can be contaminated by foreign microorganisms. Even
selection for E. coli cells with antibiotic resistance genes cannot entirely preclude
microbial contamination.
The safest way to approach work with live microorganisms is to make the following
assumptions:
1. Every microorganism used in the laboratory is potentially hazardous.
2. Every culture fluid contains potentially pathogenic organisms.
3. Every culture fluid contains potentially toxic substances.
The basis of a microbiology laboratory code of practice is that no direct contact
should be made with the experimental organisms or culture fluids, e.g. contact with
the skin, nose, eyes or mouth. As we all know from our experience with common
colds, a large proportion of infections result from the inhalation of infectious aerosols.
The same holds for laboratory-acquired infections arising from laboratory procedures.
9

10. LAB RULES
1. Prepare for each laboratory period by reading each exercise and becoming
familiar with the principles and methods involved. By being familiar with the
exercise you decrease the chances of an accident. Also, advanced preparation
allows you to use your time efficiently in the laboratory to complete the
experiment.
2. No eating, drinking, or smoking is permitted in the laboratory.
3. Laboratory coats and safety glasses must be worn at all times in the laboratory.
This is to ensure that culture material is not accidentally deposited on your
clothes or skin, and as a safeguard to protect your clothes and yourself from
chemical spills and stains.
4. Wearing gloves is mandatory during all pracs.
5. Only those materials pertinent to your laboratory work, such as laboratory
manuals, laboratory notebooks and other laboratory materials, should be
brought to your laboratory workspace. All other items, such as coats, books
and bags, should be stored away from your work area.
6. Begin each laboratory session by disinfecting your work area. Spray the area
with a disinfectant (80% ethanol), spread the disinfectant with a paper towel
and allow the area to dry. In the current pracs, this has been done by the
teaching staff for you. However, you must repeat this procedure after you have
finished your work to ensure that any material you have deposited on the work
surface is properly disinfected.
7. All material and chemicals should be properly labeled with your name, class,
date and contents. Labelling is critical to avoid improper use or disposal of
material.
8. All material contaminated with living E. coli must be disinfected or autoclaved
before disposal or reuse. There are containers for the collection of all material
to be autoclaved. Separate bins are provided for sharp waste (needles, glass
pipettes).
9. After the laboratory session, observe good hygiene by washing your hands
before leaving the laboratory.
10. Be very careful with Bunsen burners. To avoid injuries, burners should be
turned off when not in use. When reaching for objects, be careful not to place
your hands into the flame. In this set of pracs we will avoid using Bunsen
burners.
11. In the event of any accident or injury, report immediately to the laboratory
instructor so that prompt and proper action can be taken.
There is no laboratory access outside of scheduled laboratory hours and no access
without RSC staff being present.
10

11. Pre-Laboratory Preparation
1. Know your experiment: Draw an outline of the experimental procedures first by
reading the practical manual. It is important to consult your texts so you can
anticipate the outcomes. Make sure you know the significance of each step and
the reasons for the use of each reagent and technique. A brief flow chart can be of
great value.
2. Potential Hazards: You are required to complete a risk assessment for each
experiment you intend to undertake (the risk assessment forms are in the
appendix). Potential hazards such as fire, explosion, pressure development, heat
evolution, toxic gas emission and general toxicity are low for the experiments in
this course but proper protective measures must be accounted for and relevant
safeguards put into place. Before you commence any lab work your completed
risk assessment must be signed and authorised by a demonstrator. Information on
the safety and risk assessment procedures are in the Appendix of this manual. It is
a legal requirement that you are aware of the hazards associated with a particular
experiment before you commence that procedure. This includes methods for
handling and disposing of reagents and biological materials. Relevant information
is readily available through “Chemwatch” which is an online database of
chemical safety information.
3. All of these steps must be completed BEFORE you enter the laboratory.
Chemwatch access
1) Use this autologin link:
http://jr.chemwatch.net/chemwatch.web/account/autologinbyip/ , which should work
from all ANU computers.
2) If you are off-campus, use the ANU's reverse proxy procedure (choose "internal
jump point" and then paste the same web address as provided under point 1 (see
above) into the URL field, then confirm with "GO"). Alternatively, you can use this
link:
http://jr.chemwatch.net.virtual.anu.edu.au/chemwatch.web/account/autologinbyip/ ,
which is doing exactly the same.
11

12. Notebook
All students are required to maintain a laboratory notebook. The notebook will be
used for the recording of laboratory data and calculations, and will be critically
important for writing your lab reports. An experiment is not properly carried out
unless it is properly documented! A notebook should comprise all pre-laboratory
preparations and rationale as well as a record of the experiment being performed. It is
important that the notebook is dated (a notebook can potentially be a legal document).
Observations should always be written directly into the notebook as they are made.
Do not rely on memory and do not use loose pieces of paper to record your
observations. It is a good idea to make subsections in the notebook for each
experiment with the subheadings aim, methods/procedures, results. A good notebook
will allow assessment and understanding of the experimental details many years later.
Lab Report
The lab report should succinctly report the experiment performed with the
subheadings aim, methods, results, discussion. Figures and diagrams must be
numbered and must have a legend. Each figure and diagram must be referred to
explicitly in the text. Consult your favourite scientific journal for the appropriate
format.
Laboratory Etiquette
A clean and tidy laboratory makes for a safe working environment. You will be
expected to adhere to the following guidelines for laboratory maintenance.
1. All reagents should be returned to the appropriate area immediately after use.
2. Clean up spilt reagents or solvents immediately.
3. If you discover any damaged fittings (taps, electrical points, pumps, etc.)
report them at once so they can be attended to.
4. Make sure that all waste materials are disposed of into the correct waste
container. There are three basic waste types – biological waste, reagent waste
and general waste. All biological waste containing proteins and DNA goes to
the biohazard bin (tubes, tips, used gloves, etc.). Reagent waste goes to the
waste reagent container (waste reagent from the kit belongs to this category).
General waste is waste without contamination by chemicals or living
materials. Consult a demonstrator when in doubt as to where a residue should
go.
5. Any containers that have been in contact with live bacterial cells must be
treated with bleach for 24 h before disposal.
6. Do not throw anything down the sink, unless explicitly directed by your
demonstrators/technical staff.
12

13. EXPERIMENTAL PROTOCOLS
Week one
Monday
Site-directed mutagenesis
Using two mutation primers, the plasmid DNA is linearized by PCR. As always in
PCR, the mutant primers become part of the final product. After the PCR
amplification, the original plasmid is digested by DpnI, the PCR product is digested
under controlled conditions with the 3’ exonuclease E2 and recircularised via
annealing of the optimum homologous 5’ overhang sequences produced at both ends
by the exonuclease digestion. After transformation of the reaction mixture into E. coli
cells the E. coli DNA repair system will fill the gaps and seal the nicks to form fully
replicable circular plasmid.
Equipment
Bench top centrifuge
Thermocycler
Water bath set at 37°C
Heat block set at 72°C
PCR purification kit (Bioline)
NanoVue spectrophotometers
Bunsen burner
Incubator set at 37°C
Agarose gel electrophoresis system
GelDoc system
Spreader
Reagents
DNA primers (oligos)
Plasmid DNA template
50 x dNTP
5 x Q5 reaction buffer
Q5 DNA polymerase
RedSafe dye
PCR purification kit (spin columns, collection tubes, binding buffer CB, wash buffer
CW and elution buffer C)
MQ H2O (sterilised)
LBAmp plates
10 x E2 buffer (Otting lab)
E2 exonuclease (Otting lab)
DpnI restriction enzyme
Chemically competent cells (E. coli DH5α or DH10B with competency of 1 x 106
c.f.u/µg pUC19 plasmid DNA or above)
1. Vector linearisation by PCR amplification (3.5 h)
Set up PCR reaction in 0.2 ml PCR tube:
DNA template 2 µL
50 x dNTP 2 µL
5 x Q5 reaction buffer 20 µL
13

14. Forward primer (10 µM) 5 µL
Reverse primer (10 µM) 5 µL
MQ H2O (sterilised) 64 µL
Q5 (NEB, 2 U/µL, demonstrator adds) 1 µL
Total 100 µL
Mix by pipetting while looking at the solution from the side. Be careful that no visible
drop is left on the inner wall of the PCR tube. Divide into two tubes (because the
volume of the solution is larger than recommended for a single PCR vial) and run
PCR.
The PCR cycling program in a thermocycler is as follows: Initial denaturation at 98
°C for 2 min, followed by 30 cycles of (98 °C for 20 sec, 55 °C for 20 sec, 72 °C for 3
min) and final extension at 72 °C for 5 min. Take out and combine the reactions from
the two tubes into a single tube by pipetting. Mix by pipetting. (At this point, the
reaction product can be stored on ice if necessary.)
2. Agarose gel electrophoresis (1 h)
PCR reactions need to be analysed by agarose gel electrophoresis to verify that the
amplified DNA fragments have the correct size.
(1) While the PCR is running, prepare a 1% agarose gel containing RedSafe stain.
Dissolve 0.3 g agarose in 30 mL 1x TAE buffer in a 200 mL Scotch bottle or
equivalent and heat in a microwave to boiling. Cool on bench for 5 min and repeat the
boiling to dissolve all particles completely. WARNING: use protective gloves or a
thick layer of tissues to hold the hot flask to avoid burns. Once the agarose solution
has cooled to about 50 °C, add RedSafe dye (20,000x dilution, 2.5 µL in 50 mL
agarose gel solution) and mix by swirling. Insert tray into gel tank and pour agarose
gel into tray with comb in place. The gel takes about 20 min to set.
(2) Take out tray with set gel, turn 90º and insert into gel tank oriented wells side
towards negative pole. Fill tank with 1x TAE buffer just above the gel surface,
ensuring all the wells are filled with buffer. Mix 1 µL 6x loading dye into 5 µL PCR
reaction product. Load all into the gel slot alongside a lane with 10 µL DNA ladder
(see Appendix B for a description of the DNA ladder). Run the agarose gel
electrophoresis at 100-110 V for 40 min until the loading dye has migrated two-thirds
of the way to the end of the gel.
(3) Place the gel inside the GelDoc imaging chamber on UV box following
demonstrator’s instructions. If the expected PCR product produces a clear single band
proceed to section 3 for PCR purification. Otherwise a purer product must be obtained
by modification of the PCR cycling conditions or gel purification (i.e. run agarose gel
electrophoresis of the rest PCR product to separate and cut out the band with the
correct product from the agarose gel and use an extraction kit to isolate the DNA).
For the lab book: take a photo of the agarose gel.
3. PCR purification (0.5 h)
PCR reactions can be purified using a PCR purification kit (Bioline) as follows.
(1) Transfer 100 µL PCR reaction into twice the volume (i.e. 200 µL) of binding
buffer CB, mix and transfer into a spin column sitting in a collection tube and spin at
11,000 g for 30 sec.
(2) Discard flowthrough into waste container, place column back in collection tube,
14

15. add 700 µl wash buffer CW and spin at 11,000 g for 30 sec.
(3) Discard flowthrough into waste container, place column back in collection tube
and spin for an additional 1 min at 11,000 g with lid open.
(4) Place column into a clean 1.5 mL microcentrifuge tube, carefully add 30 µL
elution buffer C onto the silicon membrane of the column without touching the
membrane, leave for 1 min and spin at the same speed for 1 min.
(5) Measure DNA concentration on NanoVue spectrophotometer (see Appendix C)
4. Assembly reaction (1.5 h)
Calculate the volume of PCR product to be used and set up the assembly reaction by
adding the following in sequence:
PCR product (200 ng) x µL
10 x E2 reaction buffer 2 µL
DpnI restriction enzyme 1 µL
MQ H2O (sterilised) 20-x-4 µL
E2 exonuclease 1 µL
Total 20 µL
Add DpnI and E2 last (demonstrator does the addition), mix by pipetting and place in
37 °C water bath for 1 h. Afterwards, transfer the reaction tube into a 72 °C heat block
for 20 min to inactivate the enzymes. After heating, leave the reaction tube on bench
for 5 min to cool down slowly, then store on ice.
5. Transformation (0.5 h)
Pre-warm LBAmp plates at 37 °C, starting at the same time as the E2 reaction.
Add 10 µL of the reaction mixture to 100 µL pre-thawed chemically competent cells,
mix vigorously by ratcheting the bottom of the tube rapidly across the holes of an
empty Eppendorf tube rack 3-4 times. Quickly place back on ice for 5 min.
Light a Bunsen burner and work under the flame to transfer all cells onto a pre-
warmed 37°C LBAmp plate, spread evenly on the plate surface with a spreader to dry,
close the lid and incubate at 37°C incubator for overnight (O/N).
Tuesday
For the lab book: take a photo of the plate. Count the colonies.
The colonies appearing on the selective agar plate do not all contain the desired
mutagenized plasmid as some might also contain the original plasmid used as PCR
template. The DpnI restriction enzyme in the E2 reaction selectively digests template
DNA at methylation sites (see figure below; plasmid DNA purified from E. coli is
methylated at many sites) but not PCR-amplified DNA. Any residual parental plasmid
undigested by DpnI, however, will be transformed into cells and give rise to colonies
on the selective agar plate. DNA sequencing is therefore critical to single out the
correctly mutagenized from parental clones.
Figure: the E. coli DNA adenine methyltransferase (Dam) methylates adenine in the
15

16. N6 position at GATC sites. DpnI digests these sites.
Lab report 1:
Your lab report should contain
- A picture of the agarose gel with your DNA. Identify the size of the DNA (in
nucleotides) by comparison with the DNA ladder (see Appendix).
- A picture of the plate with the colonies grown. Report the number of colonies.
- A report of the amount of DNA obtained in the plasmid preparation (report
absorption measured by NanoVue, concentration in ng/µL and volume). Also
report the absorption ratio A260/A280 as a criterion of the purity of the DNA.
DNA Sequencing
To gain enough plasmid DNA for sequencing, a single colony needs to be picked and
amplified by growing more cells. Subsequently, the cells must be lysed and the
plasmid DNA isolated. A PCR reaction containing nucleotides with fluorescent dyes
is performed. The product is chromatographed and read by an automated DNA
sequencer (at the Biomolecular Resource Facility at the John Curtin School of
Medical Research).
Equipment and reagents
Incubator shaker set at 37°C
Bunsen burner
Lighter
10 mL culture tube
Ampicillin (100 mg/mL)
LB liquid medium
Cell culture O/N (0.5 h)
Under the Bunsen burner and using a 10 µL pipette tip sitting at the end of a 1 mL
blue tip, randomly pick two well separated medium-sized colonies and inoculate two
separate 5 mL LB liquid media supplemented with 100 µg/mL (add 5 µL 100 mg/mL
stock into 5 mL) ampicillin. Care must be taken that only a single colony is touched
by the pipette tip. Label clearly with sample number, i.e, 2-1, 2-2 for mutant 2
samples 1 and 2 respectively. Place tubes inside 37°C incubator shaker O/N.
Wednesday
Equipment and reagents
Bench top microcentrifuge
NanoVue spectrophotometer
Vacuum dryer/desicator
Qiagen plasmid mini prep kit (buffers P1, P2, N3, PE and EB)
PET3 oligo primer (1 µM)
5 x sequencing buffer
Bigdye
EDTA (125 µM, pH 8.0)
Absolute ethanol
70% ethanol
16

17. 1. Plasmid miniprep and DNA concentration determination (1 h, work in groups,
2 or 4 students per group)
(1) Transfer 1 mL of each O/N culture into separate Eppendorf tubes and spin for 1
min at top speed. Pour off supernatant into waste container, transfer another 1 mL into
the same tube and spin as before. Repeat 3 more times until all culture is spun down.
Remove as much as possible of the final residual supernatant by pipetting without
perturbing the cell pellet.
(2) Add 250 µL of resuspension buffer P1 (50 mM Tris.
HCl, pH 8, 10 mM EDTA,
100 µg/mL RNase A, LyseBlue) and mix thoroughly by pipetting up and down.
(LyseBlue is a proprietary dye, poorly soluble in buffer P1 but soluble in buffer P2. It
is blue at the pH of buffer P2. It serves as an indicator of homogeneous mixing in step
3.)
(3) Add 250 µL of lysis buffer P2 (200 mM NaOH and 1% SDS), mix by reversing 10
times and leave at room temperature for no more than 5 min. Occasionally mix by
reversing a few times if dark blue dots are seen during incubation. Do not vortex or
mix vigorously as shearing breaks genomic DNA, the sheared smaller fragments of
which would be co-purified.
(4) Add 350 µL of neutralisation buffer N3 (4.2 M guanidinium.
HCl, 900 mM KOAc,
pH 4.8) and mix by reversing 10 times. LyseBlue is colourless at this pH. Spin at top
speed for 10 min.
(5) Transfer supernatant to spin column sitting in a 2 mL collection tube and spin at
13,000 rpm (~17,900 g) for 1 min.
(6) Discard flow-through, add 700 L wash buffer PE (10 mM Tris.
HCl, pH 7.5, 80%
ethanol) and spin at 13,000 rpm (~17,900 g) for 1 min.
(7) Discard flow-through and spin for an additional 1 min to remove residual wash
solution.
(8) Place the spin column in a clean 1.5 mL Eppendorf tube and add 50 µL elution
buffer EB (10 mM Tris.
HCl, pH 8.5) onto the membrane of the column. Incubate at
room temperature for 1 min and then spin for 1 min at 13,000 rpm (~17,900 g).
(9) Measure plasmid DNA concentration using NanoVue spectrophotometer (see
Appendix C).
2. Sequencing
Calculate the volume to be used (using the concentration determined in step 9 above,
100~300 ng required per sequencing reaction) and set up the DNA sequencing
reactions as follows (volumes in µL):
Clone 1 Clone 2
DNA x y
1 µM PET3 primer 3.2 3.2
5 x buffer 4 4
BigDye (to be added by demonstrators) 1 1
H2O 20-x-8.2 20-y-8.2
total (µL) 20 20
When mixing, make sure that any drops on the inside wall of the PCR tube are
included in the mixing by pipetting up and down to ensure complete mixing of all
17

18. components.
PCR temperature cycling: 35 cycles of 96 °C for 10 sec, 50 °C for 5 sec, 60 °C for 4
min. In total the PCR reaction will take about 3.5 h. Afterwards take out the samples
and proceed to step 3 for clean up.
3. Sequencing reaction clean up and sample submission
(1) Transfer all of the 20 µL reaction into a labelled Eppendorf tube, add 5 µL 125
mM EDTA (pH 8.0) and 60 µL absolute ethanol. Close lid, mix by tapping with
finger a few times and leave on bench for 15 min.
(2) Spin at top speed (~16,000 g) for 20 min. Make sure that the plastic link between
the tube and the lid always points up so that you can guess the location of the
precipitated DNA pellet side (which is not visible). Carefully remove the supernatant
without touching the pellet area, using a 10 µL pipette tip mounted on a yellow tip.
Demonstrator: show how to do this. It is important to be quick, as the pellet easily
becomes loose or leaves from original spot over time.
(3) Fill tube with 250 µL 70% ethanol and spin for 10 min at top speed. Carefully
remove the supernatant as above. Vacuum-dry the pellet with lid open. Normally 5-10
min are sufficient.
(4) Fill out a sample submission form and submit the samples to the biomolecular
resource facility (BRF) at the John Curtin School of Medical Research. This will be
done by staff/demonstrator. Make sure each sample uses a unique ID.
Thursday
For the lab book: report the sequencing result and compare with the original
sequence. Has the mutation succeeded?
Lab report 2:
1. Report the DNA yield obtained in the plasmid miniprep (concentration and
volume).
2. Show the sequencing chromatogram for the part that includes the codons of
residues of interest. Identify the codons of these residues. Did you obtain the
desired mutation?
3. Report on the correctness (or otherwise) of your product using Clustal.
Download the graphical version (ClustalX) from
http://www.clustal.org/clustal2/.
4. Pick an example of a terminating nucleotide triphosphate with dye used in
sequencing from patent EP1546391 A2. Depict its structure.
Equipment and reagents
Incubator set at 37 °C
Chemically competent XJb(DE3) Autolysis cells (Zymoresearch)
1. Sequence alignment analysis
There are many sequence alignment programs available online for free. CLUSTALW
is an example for this analysis. Enter the http://www.genome.jp/tools/clustalw/ web
page. The screen shots below describe the process step by step.
ClustalX is better, but needs to be downloaded onto your computer
(http://www.clustal.org/clustal2/). It also provides access to the chromatogram, but
18

19. requires Java to do so.
Figure 1 shows how to enter (by copy and paste) the nucleotide sequences of your
expected and actual (sequenced) clones. Each sequence needs a header line starting
with a “>” symbol. In the example below, “>DCLRS” is the expected sequence and
“>14105” is one of the sequenced clones. The entered sequences are shown in the
following lines. When all is done, click “execute multiple alignment” to obtain the
aligned sequences. All correctly aligned bases are marked as “*” while incorrect ones
are left as empty spaces. Therefore, simply by checking for the empty spaces you will
know how good your sequence is. If there is no empty space from beginning to end,
your sequence is 100% correct. Figure 2 shows a correct sequence and Figure 3 shows
a sequence with a single nucleotide change.
Figure 1. Screenshot 1
19

20. Figure 2. Screenshot 2
Figure 3. Screenshot 3
This is a G to T change. When this sort of inconsistency happens, it is important to
check whether it’s a genuine error, as the sequencing profile is nowadays read
automatically and the computer could have made a reading error. This can be clarified
by visual inspection of the original profile at the specific region. If confirmed as a true
error, it may still not change the amino acid sequence due to the degeneracies in the
genetic code. In the example above, GAT (D) is changed to TAT (Y), which is not
acceptable inside the open reading frame.
20

21. In vivo expression of a mutant RS enzyme
In vivo expression starts with the transformation of an expression strain. The strain
“XJb(DE3) Autolysis” has the T7 RNA polymerase gene in its genome. The
polymerase expression is under control of the lac operon. IPTG induction releases the
lac repressor, leading to expression of the T7 RNA polymerase, leading to the
transcription and translation of the target protein (in this case the mutant RS enzyme).
Prior to induction, a sufficient number of E. coli cells at vigorous physiological state
must be grown. This is done in two steps: first a small-scale (2 mL) starter culture is
grown, which is then used to inoculate the large-scale (100 mL) culture. The large-
scale culture is induced with IPTG, when the cells are in the exponential growth
phase.
2. Transformation
Each student is to transform plasmid from a sequence-verified clone into the protein
expression host cells XJb(DE3) Autolysis by the quick method as on Day 1. Mix 1 µL
of the plasmid with 100 µL chemically competent XJb(DE3) Autolysis cells, incubate
on ice for 5 min, then plate out on pre-warmed 37 °C LBAmp plate. Incubate the plate
in 37 °C incubator O/N.
Friday
Equipment and reagents
Incubator set at 37 °C
Inoculation of start culture for in vivo protein expression
Under the Bunsen burner flame, use a yellow pipette tip to touch 3-5 well-separated
medium-sized colonies from the transformation plate and inoculate 2 mL LBAmp (2 µL
100 mg/mL Ampicillin in 2 mL LB) liquid medium in a 10 mL culture tube. Store the
culture tubes in fridge (~4 °C). Staff will move the culture tubes on Sunday afternoon
into a 37 °C incubator to culture O/N.
Week 2
Monday
Equipment and reagents
Incubator shaker
Centrifuge with 50 mL Falcon tube rotor
Visual spectrophotometer
250 mL baffled flasks
20% arabinose
0.5 M IPTG
Protein overexpression by IPTG induction
(1) Vigorously shake the O/N culture to obtain a homogeneous cell suspension.
Inoculate 2 mL of the O/N culture into 100 mL LBAmp liquid medium and grow in 37
21

22. °C incubator shaker until the OD600 reaches 0.7-1.0. This requires monitoring the cell
growth by transferring 1 mL culture into a disposable cuvette to measure the OD600.
Make first measurement after 3 hours and second measurement at time interval you
calculated. XJb(DE3) cell doubling time at 37 °C is about 50 min, i.e. you can
estimate how long it will take from the first measured point that you made to the
required induction point OD600 0.7-1.0.
(2) When the OD reaches the required level, put 1 mL of culture aside in an
Eppendorf tube as the “before induction” sample. Add 200 µL 0.5 M IPTG and 225
µL 20% arabinose into the culture and continue shaking for O/N at room temperature.
(3) This step has to be done by staff/demonstrator as no undergraduate student access
to the centrifuge. Take out 0.5 mL of the culture as the “after induction” sample day
after in the morning. Transfer 50 mL of the culture into a Falcon tube and spin at
room temperature (RT) for 15 min. Discard the supernatant into waste, refill with the
rest of the culture and spin as before. Discard the supernatant and take out residual
liquid with a pipette without disturbing the cell pellet. Freeze the cell pellet and tube
in liquid nitrogen and store in -20 °C freezer.
Tuesday
Protein purification
The XJb(DE3) Autolysis strain constitutively expresses bacteriophage λ endolysin,
which is a protein allowing facile cell lysis by a single freeze-thaw cycle. If viscosity
is a problem, short time sonication can be used to aid the lysis. The cell debris is spun
down. The RS enzyme is soluble and it contains a His6-tag for affinity purification on
an IMAC column. The success of protein production and purification is checked by
SDS-PAGE. For our purpose, the enzyme does not need to be 100% pure, because we
will subsequently use it in a cell-free protein synthesis reaction to try and incorporate
a phosphotyrosine analogue into a GFP mutant containing an amber stop.
Equipment and reagents
Bench-top centrifuge
SDS-PAGE electrophoresis apparatus
12% Precast gels (Genscript)
His GraviTrap column (one per student)
Water bath (room temperature)
MOPS running buffer
Buffers A and B
Lysis buffer
1. Protein purification
(1) Prepare the His GraviTrap column by cutting off its end tip, remove the top cap,
pour off excess liquid and clamp the column on a stand with clamp. Note: do not cut
off the end tip when using a previously used (i.e. regenerated) column. Wash and
equilibrate first with 3 x 10 mL MQ water (the frits protect the column from running
dry during the run) and then with 10 mL binding buffer (20 mM HEPES buffer pH
7.5, 500 mM sodium chloride, 20 mM imidazole). Collect flow-through liquid in a
beaker.
(2) Take out frozen sample pellet and resuspend in 5 mL lysis buffer (20 mM HEPES,
22

23. pH 7.5, 500 mM NaCl, 20 mM imidazole, 200 µg/mL hen egg white lysozyme, 10
mM spermidine, 1 mM AEBSF) by flushing buffer onto the frozen pellet using a
plastic transfer pipette. Warm up the tube in room-temperature water-bath for 2 min
with occasional reversing with lid tightly closed. Put the sample tube on ice for 10
min. Transfer into 4 Eppendorf tubes in 4 equal volumes using a plastic transfer
pipette and spin at top speed for 5 min. Carefully transfer the supernatant into a 10
mL tube without disturbing the pellet. Sacrifice some supernatant to save only the
clear supernatant.
(3) Set aside 20 µL for SDS-PAGE analysis on ice (“before loading” sample) and
proceed with His GraviTrap 1 mL kit (GE) protein purification as follows.
(3.1) Load the supernatant (step 2) onto the equilibrated column, collect the flow-
through and reload. Repeat 3 times. Set aside 20 µL of the last flow-through for later
SDS-PAGE analysis (“flow-through” sample).
(3.2) Wash with 10 mL binding buffer. Set aside 20 µL for SDS-PAGE analysis
(“wash” sample).
(3.3) Elute protein into a new tube by applying 3 mL elution buffer (20 mM HEPES
buffer, pH 7.5, 500 mM sodium chloride, 500 mM imidazole), set aside 20 µL for
SDS-PAGE analysis (“elution” sample) and immediately hand in rest of the elution
sample to demonstrator to keep in ice and be concentrated/buffer exchanged later.
2. SDS-PAGE gel electrophoresis
Precast SDS-PAGE gels are used in this prac. Assembly, sample loading and running
of the SDS-PAGE gel is described in detail in Appendix E.
(5) Concentrating the protein and concentration determination by NanoVue
This step has to be done by staff/demonstrator as no undergraduate student access to
the centrifuge.
Transfer all eluted protein (3 mL) into a Millipore Ultra-4 10 kDa MWCO centrifugal
filter unit and spin at 4000 g for 30 min at 4 °C. Take out the filter from the collection
tube, empty the flow-through into a 10 mL Falcon tube and place the filter back into
the collection tube. Add 4 mL buffer (20 mM HEPES, pH 7.5, 1 mM DTT) and spin
at 4000 g for 20 min at 4 °C. Empty the flow-through as before and add 4 mL buffer.
Spin as before and carefully transfer the concentrated protein sample into an
Eppendorf tube (~100 µL final volume, about 30 x concentrated).
Determine the protein concentration on a NanoVue spectrophotometer (see Appendix
C).
Lab report 3:
Print an image of your SDS-PAGE result. Indicate the molecular masses of the
molecular weight markers. Estimate the purity of the mutant you have made (in
percent). Report the amount of protein made and its concentration as determined by
UV absorption. Use ExPASy (ProtParam) to calculate the extinction coefficient ε280 of
your mutant. Report the predicted extinction coefficient.
Wednesday
23

24. Cell-free protein synthesis
Cell-free protein synthesis (CFPS) uses a cell extract to make proteins. In our case,
the cell extract is a so-called S30 extract from E. coli. We use a dialysis system,
where the reaction mixture is separated by a dialysis bag from an outside mixture that
provides a source of ATP, amino acids, nucleotides and buffer, while diluting low-
molecular weight products that could inhibit the cell-free reaction (e.g. phosphate).
The aim in this practical is to explore, whether the RS enzyme that you have made in
vivo can incorporate an amino acid at the amber stop codon of the GFP gene.
Successful genetic encoding of tyrosine phosphate would allow detailed activity
studies of proteins with multiple phosphorylation sites.
Equipment and reagents
Back-and-forth motion water bath shaker set at 30 °C (Otting group)
pH meter
Scissors
Cell free protein synthesis reaction set up
1. Prepare the Reaction mixture (= Inner solution) and the Outside buffer according to
the tables below in sequence. IMPORTANT: the preparation of the amino-acid
mixture from the water-, acid- and base-soluble mixtures (see table below) must be on
the bench at room temperature (not on ice, as precipitate may form).
Volume of reaction 2 x 200 µL
amino-acid mix Volume/µL
50 mM water-soluble aa each (15 mM) 94.5
50 mM acid-soluble aa each (15 mM) 94.5
50 mM base-soluble aa each (15 mM) 94.5
milli-Q water 31.5
TOTAL 315.0
10x reaction mix
25 mM rNTP each (0.8 mM) 154.6
2.0 M HEPES (55 mM) 132.8
96 mM ATP (1.2 mM) 60.4
10 mM folinic acid (68 uM) 32.8
100 mM cyclic AMP (0.64 mM) 30.9
500 mM DTT (1.7 mM) 16.4
9.2 M NH4OAc (27.5 mM) 14.4
milli-Q water (sterile) 40.6
TOTAL (used for Inner + Outer buffers) 483.0
master mix Inner Outer
10x reaction mix 44.0 400
1 M creatine phosphate (CP; 80 mM) 35.2 320
amino-acid mix; 15 mM each (1 mM) 29.3 266.7
4 M KGlu (208 mM) 22.9 208.0
1.07 M Mg(OAc)2 (15 mM Inner; 19.3 mM Outer buffer) 6.2 72.1
17.5 mg/mL tRNA (0.175 mg/mL) 4.4 0
24

25. 10 mg/mL creatine kinase (250 µg/mL) 11.0 0
TOTAL 153.0 1266.8
Making the Reaction mixture:
+UAA (µL) -UAA (µL)
Master mix 69.5 69.5
GFP Amber construct DNA x x
S30 extract 40 40
Amber suppressor tRNA y
Purified RS (30 µM final) z z
milli-Q water (sterile) 200-x-y-z-69.5-40 200-x-z-69.5-40
TOTAL 200.0 200.0
Making the Outer buffer:
Add milli-Q water to the Outer buffer until the volume is 10x that of the Reaction
mixture (if the Reaction mixture is 200 µL, the Outer buffer must be 2 mL). Add H2O
to about 2 ml and adjust the pH of the Outer buffer to 7.5 with 1 N KOH using P20
pipette. As the pH is very close to 7.5 therefore only a few drops of 1 N KOH is
required to achieve required pH value. Use P20 pipet and yellow tips for the
adjustment.
2. Place the Reaction mixture (200 µL per sample) into Spectrapor #2 dialysis tubing
(MWCO 12-14,000) and submerge it in 2 mL Outside buffer in a 10 mL Falcon tube.
Cut the dialysis tubing into a 8-10 cm long segment and soak in MQ water. Tie a knot
at one end of the dialysis tubing and fit the other (open) end onto a 0.5 mL tube with
the end opposite the lid cut off. Open the lid, transfer the Reaction mix into the
dialysis tubing through the tube opening and put the lid back. WARNING: tying the
knots is tricky – don’t break the tubing! Place the whole dialysis tube device inside
the Falcon tube containing 2 mL Outer buffer, close the lid tightly and place in a rack.
A demonstrator will place all inside a 30 °C water bath with gentle shaking (150 rpm
in back-and-forth mode) for 16 h. Be sure that all of the Reaction mixture in the
tubing is covered by Outer buffer. A little push from the top of the Falcon tube may
do the trick.
Thursday
Analysis of protein size and yield by SDS-PAGE
Visually compare the colour of the two reactions - is there any difference? Full-length
GFP is strongly fluorescent, i.e. fluorescence intensity is an indication of protein
yield. SDS-PAGE tells whether the molecular mass is right and gives an indication of
protein yields.
Equipment and reagents
SAS_PAGE electrophoresis system
Shaker
BioSafe protein stain
Lunch box for gel staining and destaining
Precision plus protein standards
SDS-PAGE gel electrophoresis
25

26. Take out the dialysis device, cut the dialysis tubing with scissors just below the cut
Eppendorf, holding the dialysis tubing at the knot. Mix well by pipetting up and down
and transfer the Reaction mix into an Eppendorf tube. Take 20 µL of both reactions
and mix with an equal volume of 2x protein loading dye. Heat at 95 °C for 5 min to
denature all protein.
Run the electrophoresis and stain as described in Appendix E.
Lab report 4:
Photo of the cell-free reaction mixture before and after expression. Is there evidence
for fluorescence, indicating that GFP is present?
Photo of the SDS-PAGE gel. Identify the expected molecular masses of the full-
length protein and of the truncated product (truncated at the amber stop codon).
In addition, make a one-page overview summarizing the results of the entire prac,
containing:
- the date
- the number of your mutant and the residues at positions 32, 65, 103, 108, 109, 158
and 162.
- picture of the chromatogram of the sequencing result spanning residues 103-109
- picture of Tiffany’s summary SDS-PAGE showing the purified RS enzymes
- picture of the SDS-PAGE gel of the cell-free reaction.
26

27. Appendix A: Working under sterile conditions
Adapted from http://bitesizebio.com/6630/how-good-is-your-sterile-technique/
Wearing latex or nitrile gloves serves two purposes: to protect you from what you’re
working with, and to protect what you’re working with from you. The majority of
gloves sold for lab use are not sterile, so keep in mind that wearing gloves while
working with sterile materials is not a guarantee of cleanliness. Keep in mind that
gloves are designed as “single use” only, so make sure to discard them and put on a
fresh pair after taking a break from the bench. If you get bacteria on your hands, it’s a
good idea to spray them with ethanol to eliminate the majority of possible
contaminants. Here’s a great tip though – after using ethanol, be sure to let your hands
dry before lighting your Bunsen burner!
For sterile work at the bench, a Bunsen burner is your best friend. The flame is used
to directly sterilize glass bottles, spreaders, and other tools. The other major function
of the Bunsen burner is to create an updraft in the local area. Hot air rises so the
heated air around a lit Bunsen burner creates a slight current upwards. This means that
any “hovering” contaminants in the air are wafted away from your work area, instead
of falling into your work. The flame sterilizes not only the air above it but (by air
circulation) the space anywhere within 20 cm of the flame. Wait 20 seconds after
lighting the flame to allow sterile conditions to build up. While it’s important to work
near the flame, don’t get so anxious about it that you burn yourself. Close is good
enough; the radius of the sterile area is about 20 cm and up to 50 cm.
Sterile materials should only be opened near a lit Bunsen burner or in a biological
safety cabinet (often called a “hood”, but not the same as a chemical fume hood).
When removing tubes, petri dishes, or other sterile plastic-ware from plastic
packaging, try to touch only the items you are removing, and reseal the packaging
immediately to keep the remaining items from contamination. Keep your tip box
closed when not in use, for the same reason. When using media in glass containers,
run the mouth of the bottle through the flame of your Bunsen burner before inserting a
pipet and before replacing the lid. This helps to prevent contamination from the lip of
the bottle. This cannot be done with plastic bottles (at the risk of melting). Use them
near a flame and try to avoid touching the mouth of the bottle with your pipet.
27

28. Appendix B: NEB 2-log DNA ladder (0.1 – 10.0 kb)
The DNA ladder contains a number of proprietary plasmids that have been digested to
completion with appropriate restriction enzymes to yield 19 bands suitable for use as
molecular weight standards for agarose gel electrophoresis. This digested DNA
includes fragments ranging from 100 bp to 10 kb.
Figure: 2-Log DNA ladder visualized by ethidium bromide staining on a 1.0% TBE
agarose gel. The 0.5, 1.0 and 3.0 kb bands have increased intensity to serve as
reference points. The column on the left provides the approximate mass of DNA in
each of the bands (assuming a 1.0 μg load). This allows estimating the mass of DNA
in comparably intense bands of similar size.
28

29. Appendix C: DNA concentration determination by NanoVue
Figure 1 shows the NanoVue instrument and the application of a sample for
measurement. In the DNA mode, the dilution factor should be set to 1.000, the display
units to ng/µL and the background correction should be on. Pipet 2 µL elution buffer
onto the black spot between the four alignment spots on the sample plate, then press
the 0A/100%T key to zero the buffer reference. Lift the sampling head and clean the
top and bottom plates with a tissue. Pipet 2 µL of the DNA solution onto the sample
plate, taking care not to introduce bubbles into the sample, gently lower the sample
head onto the sample without pushing, then press measure (A cuvette plus arrow key)
button. Record the DNA concentration and the 260/280 ratio (i.e. the ratio of
absorption at 260 and 280 nm wavelength), as this is an indication of sample purity:
pure DNA preparations have expected ratios of 1.7-1.9.
Figure 1. NanoVue UV/Vis spectrophotometer and loading with sample.
29

30. Appendix D: Protein purification using a His GraviTrap column
The figure below shows the steps for protein purification using a His GraviTrap
column. The column contains Sepharose modified with NTA and precharged with
Ni2+
.
1. Cut off the bottom tip, remove the top cap, pour off excess liquid and fasten
the column to a stand.
2. Equilibrate the column with 10 mL binding buffer. The frits protect the
column from running dry during the run.
3. Add the sample. A volume of 0.5-35 mL is recommended. The protein binding
capacity of the column is high, approx. 40 mg histidine-tagged protein/column
(protein-dependent).
4. Wash with 10 mL binding buffer.
5. Apply 3 mL elution buffer and collect the eluate.
The columns are expensive, but can be re-used. Return used columns to demonstrator
for regeneration.
30

31. Appendix E: SDS-PAGE
(1) Assembly of the SDS-PAGE apparatus
Fig. Mini-PROTEAN 3 assembly.
The figure illustrates the gel assembly process.
1) Assemble the gels on the electrode assembly module. Two groups share one
assembly.
2) Set the clamping frame to the open position on a clean flat surface (a).
3) Place the first gel sandwich onto the gel supports at the bottom of the clamping
frame (b), with the short plate facing towards the frame. First the sandwich is tilted at
an angle of 30 degrees and subsequently gently pressed onto the clamping frame (b
and c).
4) Repeat for the second gel sandwich and close the clamping frame (d).
5) Place the assembly in the gel tank at the place where the raised plastic tabs are (e).
Align the black electrode with the black colour indicator (and red with red).
6) Fill the inserted gel assembly with 1 x electrophoresis buffer to just under the edge
of the outer plate. Check for leaks.
7) Carefully remove the comb.
8) Load 10 µL of sample into well in the following order: lysate, washing flow-
through fraction, elution fraction.
9) Fill tank half with 1x electrophoresis buffer.
10) Place the lid on top of the tank. Watch the orientation: align colour-coded banana
plugs with matching jacks. The raised tabs on each side of the tank will slide through
31

32. the corresponding slots in the lid if assembled correctly.
(2) Running the SDS-PAGE gel
Connect the electricity leads (positive to positive and negative to negative). Keep in
mind that proteins migrate always from negative (cathode) to positive (anode) in the
SDS-PAGE system. Wrong connection of the leads to the power supply will lead to
loss of your samples. Attach the power source to your gel box, adjust to 200 V and
run for about 1 h until the tracking dye has just arrived at the end of the gel.
(3) Staining and destaining of the gel
1) Turn the power off and disconnect the leads.
2) Remove the assembly and pour off the electrophoresis buffer.
3) Open the arms of the assembly and remove gel cassettes.
4) Separate the spacer plate and the small plate with the green plastic wedge.
5) Gently lift the gel with your fingers and transfer into a container filled with
MQ water. Rinse briefly and repeat once. Fill with MQ water about 1.5 cm
deep, agitate on a shaker for 10 min. Repeat washing with fresh water for
another time.
6) Decant as much of the water as possible (carefully hold the gel at the side of
the container) and fill in staining solution just enough to cover the gels. Shake
on shaker until the blue protein bands develop (about 30 min).
7) Decant the staining solution and fill MQ water into the container. Agitate as
before on shaker. Change water after 30 min and shake until the background is
clear.
(4) Scanning/photographing of the gel
Once the gel is destained it can be placed between two overhead sheets and scanned
or photographed to produce TIFF or JPEG files. Make sure no air bubbles are trapped.
(5) Estimating molecular mass
The molecular masses of the various proteins can be estimated by comparison with
the protein markers shown in the Appendix below.
32

33. Appendix F: BioRad protein molecular weight markers (10 – 250 kDa)
The standards consist of 10 Strep-tagged recombinant proteins.
Figure: protein markers visualized by Comassie Blue staining on an SDS-PAGE gel.
The three proteins with MW = 25, 50, 75 kDa are at higher concentration to serve as
reference bands. The molecular mass (in kDa) is indicated for each band.
33

34. Appendix G: Making amino acid mixtures: (for 10 mL of each mixture)
A. Water soluble
amino acid weight (mg)
Ala 44.5
Arg 105.0
Gly 37.5
His 105.0
Lys 91.3
Pro 57.6
Ser 52.6
Thr 59.6
Val 58.6
B. Acid soluble (in 1 N HCl)
amino acid weight (mg)
Asn 66.0
Asp 66.5
Cys 60.1
Glu 73.5
Gln 73.1
15N
Leu 131.2
Met 74.6
Trp 102.0
Tyr 90.6
C. Alkaline soluble (in 1 N KOH)
amino acid weight (mg)
Ile 65.5
Phe 82.5
34

35. APPENDIX
Chemical Risk Assessment
• General
The “Chemical Hazard Sheet” is a risk assessment of the hazards associated with one process or
procedure performed in your area. As such it is designed to make you think and be aware of the risks
associated with your work. It is also a legal requirement that a risk assessment be completed before an
experiment or procedure is started. The assessment should also include information on the disposal of
residues generated in the experiment.
These sheets should form a series that is kept in a folder in your laboratory and be readily
accessible as required. A copy of the sheet should be available within the vicinity of the activity it
describes, while that activity is in progress. This information could be used in an emergency and
so could save your life, so the more detail the better!
• Reference No.
This is for your own records so you can keep track of your Hazard Sheets. Your lab book, where
the experiment is actually written up, should refer directly to the Hazard Sheet.
• Process or Procedure
This area is used to describe the experiment and should clearly state what you are doing and on
what scale. Appropriate information includes all reagents, solvents, likely products, the amounts
used, conditions and any special techniques that have OH&S concerns (e.g. UV radiation or high
pressure equipment). A diagram of the experimental setup may be useful if it is particularly
complex.
• Chemwatch Hazard Ratings
The Chemwatch database gives a rating on chemicals based on five criteria. Flammability – how
much of a fire risk, Toxicity – how poisonous, Body Contact – how damaging to the skin,
Reactivity – how dangerous when mixed with other compounds, Chronic – poisoning effects
which may take time to manifest themselves (e.g. cancer).
Where possible a Chemwatch search should be done on all the reagents and solvents involved.
When a compound is not listed, a chemically similar reagent should be sought and it’s ratings
used.
From this search it should be obvious what the major areas for concern are. If a reagent has a
rating of “High” or “Extreme” then precautions must be taken. These precautions include disposal
procedures.
• Hazards and Precautions
Accompanying the hazard ratings is a series of statements (Risk and Safety statements) that
provide specific information on the nature of the associated risk. The more pertinent of these
statements should be recorded especially when associated with a High or Extreme hazard rating.
From these statements it is up to the experimenter to decide on the appropriate precautions to
be taken. These precautions should be noted in the space provided on the sheet. Also note any
scheduled poisons used or generated in the experiment.
• Emergency/Disposal Action
The action to be taken in the event of an emergency should be spelt out in this space. It is best to
plan for the worst-case scenario. The most life threatening hazard should be dealt with first, as
often many hazards are associated with a chemical process. How well this is planned could mean
the difference between your own life and death! All disposal procedures should be listed here
also. In particular methods for rendering explosive or highly reactive substances harmless must
be included. All waste containers must be clearly labeled with the major (i.e. >10%) components
noted on the label. All highly reactive substances must be similarly listed on the residue bottle
regardless of concentration. It is a University OH&S requirement that a stability risk assessment
(low, medium, high risk) be performed on each residue container whenever new components are
35

36. added. This may result in an upgrading of the risk. No new components should be added to a
container already listed as “high risk”.
•RSC Risk Assessment Category
The staff and research students of the RSC have to undertake a slightly different risk assessment
schedule than those undertaken by undergraduate students. In preparation for 3rd
year students
taking on CHEM3060 projects, you should access the RSC safety regulations at
http://rsc.anu.edu.au/internal/index.php?
option=com_content&view=article&id=82&Itemid=131 or see the printed copy of the RSC safety
regulations in lab, and undertake this part of the risk assessment also. The sections on “Process
or Procedure” and “Dangerous Goods” on the Risk Assessment Sheet will help you with this.
• Validation
Your name along with contact details and date should be filled in. For inexperienced investigators,
especially Honours students, your supervisors’ signature is required in order to validate your
assessment. Supervisors’ check boxes are provided in order to ensure that aspects such as chemical
incompatibilities, residue disposal and poison scheduling have been considered. The risk assessment is
not considered valid until these boxes have been checked and the assessment signed.
• Dangerous Goods
All chemicals used should be listed under the appropriate class (NB – some chemicals fall under two
classes). This is an important aid when deciding the correct action for the disposal of residues. All
residue containers should be labeled with the appropriate DG class sticker. Chemwatch provides
information on DG classes.
36

37. CHEM3204 Risk Assessment Sheet
Process or Procedure {include all chemicals used/produced, and amounts used}
Biological Risk Rating: E. coli laboratory strains DH10B and XJb(DE3) are used
throughout this laboratory course. Although these E. coli strains are of low individual
and community risk and considered to be low risk, the standard microbiological
laboratory procedures and guidelines must be followed.
Chemwatch Hazard Ratings {min, low, moderate, high, extreme}
Reagent or Solvent Flammability Toxicity Body Contact Reactivity Chronic
Hazards {R and S statements} Precautions
Emergency/disposal action:
Risk Level as determined by RSC Research Division risk assessment protocol, section
6.2, at (http://rsc.anu.edu.au/internal/index.php?
option=com_content&task=view&id=19&Itemid=37 ):
Category (please tick √) A ☐ B1☐ B2☐ C☐
Name: Phone: Date:
Supervisors check list {have the following issues been considered}
Chemical Incompatibilities ☐ Residue Disposal ☐ Poison Scheduling ☐
Authorized by: Signature:
37

38. Dangerous Goods Classes {indicate
the DG class of all chemicals and residues involved}
Explosive Substances
Flammable Liquids
Flammable Solids
Spontaneously
Combustibles
Dangerous when wet
Oxidizing Agents
38

39. Organic Peroxides
Poisonous Substances
Corrosive Substances
Miscellaneous
Dangerous Goods
39