程序代写代做代考 Bioinformatics asp scheme DNA chain Excel BIOL5373M Protein Engineering Laboratory Project 2015-16 1
BIOL5373M Protein Engineering Laboratory Project 2015-16 1
BIOL5373M: Protein Engineering Laboratory Project
2016-17
CONTENT
Module Details 1
Module Aims 2
Learning Outcomes 2
Module Outline 2
Teaching and Learning Methods 3
Teaching Staff 3
Reading List 3
Module Timetable 8
Assessment and Assessment Deadlines 9
Information on Plagiarism 10
Laboratory Safety Information 10
Protocols 1-11 (including pre-practical questions) 11-63
Appendix 1 – Guidelines for maintaining a laboratory notebook
Appendix 2 – Researching mutants in GFP and designing primers
64-68
69-70
MODULE DETAILS
Module Code: BIOL5373M
Title: Protein Engineering Laboratory Project
Credits: 15
Semester: 1 and 2
Module Manager: Dr Chi Trinh
Room: Astbury 9.108g Phone: 0113 343 3023 Email: c.h.trinh@leeds.ac.uk
Further module information and supporting learning resources are available on the VLE.
mailto:c.h.trinh@leeds.ac.uk
BIOL5373M Protein Engineering Laboratory Project 2015-16 2
MODULE AIMS
The aims of this module are:
x To provide practical skills training in a range of modern biological techniques principally
based around molecular biology.
x To provide training in maintaining laboratory records and in writing scientific research
papers.
LEARNING OUTCOMES
On completion of this module, students should be able to:
x Demonstrate a high-level understanding of and practical competence in a range of
experimental techniques that underpin modern molecular life sciences including: plasmid
DNA preparation and quantification; subcloning; PCR; site directed mutagenesis; DNA
sequencing; recombinant protein expression; SDS PAGE and western blotting; protein
purification and analysis.
x Maintain a detailed and accurate laboratory notebook recording the experimental
procedures and results obtained during the practical sessions.
x Present and critically analyse the results obtained in the form of a short scientific paper.
MODULE OUTLINE
This module is an extended practical investigation in the form of a mini-project. The aim is to
sub-clone the gfp (green fluorescent protein) coding sequence into an expression vector,
express the protein, purify it and then analyse some of its properties. To achieve this, in pairs,
you will work through the following experimental stages:
1. Sub-clone the coding sequence of gfp DNA from pET23 into the pET28c protein
expression vector (practicals 1-3). Plasmid maps for pET23 and pET28c are included in
Figure 1.
2. Transform the plasmid into E. coli DH5D cells and select by plating on LB medium
containing kanamycin (antibiotic) followed by direct colony PCR to detect the presence
of the gfp insert (practicals 3-4).
3. Generate 2 spectral variants (mutations) of gfp using site-directed mutagenesis and
characterize by DNA-sequencing. Overall, this will yield 3 pET28c recombinants, one
carrying the wild-type gfp construct and the other two carrying (different) mutated gfp
constructs (practicals 5-6).
4. Isolate plasmid DNA derived from each of these recombinants and transform into the
expression host BL21(DE3) (practicals 6-7).
5. Induce GFP protein expression (including mutants) using the auto-induction method and
assess protein expression by SDS-polyacrylamide gel electrophoresis followed by
western blotting, using HisprobeTM-HRP (practicals 8-9).
6. Purify the GFP proteins using Ni-NTA chromatography (practical 10).
7. Analyse the purified proteins by mass spectrometry and fluorometric analysis (this will be
done for you and the data returned to you to analyse: practical 11).
BIOL5373M Protein Engineering Laboratory Project 2015-16 3
An overview of this mini-project is summarized in Table 1.
TEACHING AND LEARNING METHODS
This module will be delivered by laboratory training and experimental practice over a weekly (12
weeks) daylong sessions (with the exception of practical 3 where the experiment is split over
two half days). You will be required to keep an individual contemporaneous record of
procedures and results in the form of a laboratory notebook, which should be available for
inspection by the module teaching staff during every practical session. The results of the
investigation will be written-up in the form of a short research paper (in the style of FEBS
Letters) at the end of the practical.
You are expected to come to each practical having read the protocol of the week and the
preparatory (background) reading associated with each practical. This preparatory
reading will include some questions intended to assist you in your understanding. These should
be completed prior to attending the classes. The demonstrators will go through the answers
during your practical session. End of module test questions will be derived from these.
This module will utilize and develop skills introduced in the Advanced Biomolecular
Technologies module. Therefore, the lectures and seminars delivered as part of Advanced
Biomolecular Technologies form part of the essential background material to this practical
module and have been deliberately timetabled to precede the practical skills you will be using.
For an overview, see Table 1.
TEACHING STAFF
This module will be led by Dr Chi Trinh, supported by Dr Patrick Murphy along with the
laboratory technical staff and demonstrators. Some specialist equipment training will take place
in specialist research labs led by research leaders (e.g. mass spectrometry). If you have any
queries relating to this module, please contact Dr Trinh.
READING LIST
The companion textbook to the techniques covered in this module (and in BIOL5272M
Advanced Biomolecular Technologies module) is:
Divan, A & Royds, J. (2013) Tools and Techniques in Biomolecular Science. Oxford University
Press. Oxford.
If you are unfamiliar with the basic principles of gene cloning, then the textbook below will be
useful:
Brown, T.A. (2010). Gene Cloning and DNA Analysis. 6th Edn. Blackwell Publishing.
For an overview of recombinant protein production, refer to the following:
Zerbs, S., Frank, A.M., Colart, F.R. (2009). Bacterial systems for production of heterologous
proteins. Methods in Enzymology, 463, 149-168.
Divan, A & Royds, J. (2013) Tools and Techniques in Biomolecular Science. Oxford University
Press. Oxford. CHAPTER 7 (sections 7.1-7.3).
BIOL5373M Protein Engineering Laboratory Project 2015-16 4
For background to GFP structure, properties and uses:
Tisein, R. (1998). The green fluorescent protein. Annual Reviews of Biochemistry 67, 509-
5044.
Additional references are included on the VLE.
BIOL5373M Protein Engineering Laboratory Project 2015-16 5
Figure 1a: Novagen pET23 plasmid map
pET23gfpuv was constructed by inserting the open reading frame of gfpuv between the EcoRI
and HindIII sites of pET23. An NdeI site was also introduced at the gfpuv start codon
(underlined). Subsequently an internal NdeI site was removed by site directed mutagenesis.
Note: plasmid size is 3592bp without the gfpuv coding sequence inserted.
gfpuv open reading frame
HindIII
Nde I
EcoRI
CATATG……………………………………………………………………………TAA…
Nde I
761 bp
BIOL5373M Protein Engineering Laboratory Project 2015-16 6
Figure 1b: Novagen pET-28a-c(+) expression vector. The vector carries an N-terminal
HisxTag£/thrombin/T7xTag£ configuration plus an optional C-terminal HisxTag sequence. The
cloning /expression region of the coding strand transcribed T7 RNA polymerase is shown below
the circle map.
Table 1: Overview of the Project
Process Content Specific technology you
will learn
Associated reading
(from lectures/
workshops – part of
BIOL5270M &
BIOL5272M )
Sub-clone gfpuv
gene from the
plasmid pET23 into
the pET28c
expression vector
(practicals 1-4)
1. Measuring the DNA concentration of pET23-gfpuv
plasmid DNA
2. Digest plasmid pET23-gfpuv with restriction enzymes
NdeI and HindIII
3. Excise gfpuv band from agarose gel
4. Extract gfpuv DNA from gel and quantify
5. Ligate gfpuv insert with vector pET28c
6. Transform ligation products into DH5α cells
7. Identify the positive clones by colony PCR
UV spectroscopy/DNA
quantification
Setting up restriction
digests
Agarose gel electrophoresis
DNA gel extraction
Ligation
Transformation
PCR
Introductory lecture
(BIOL5273M)
PCR lecture
Introduce mutations
within the gfpuv
gene and identify
mutations generated
(practicals 5-7)
1. Create mutations within the gfpuv insert cloned into
pET28c using site-directed mutagenesis (SDM)
2. Transform SDM products into competent XL-1 blue cells
and culture positive clones
3. Extract plasmid pET28c DNA and check its purity and
concentration
4. Sequence the DNA and analyse sequencing data
Designing primers for site-
directed mutagenesis
Site-directed mutagenesis
Transformation
Plasmid purification
DNA sequence analysis
DNA mutagenesis
lecture
DNA sequencing lecture
BIOL5270M practical
bioinformatics
workshops
Induce protein
expression
(practicals 8-9)
1. Transform pET28c vector carrying gfpuv (or mutant)
insert into the E. coli expression host, BL21(DE3) cells
2. Identify and culture positive clones
3. Induce GFPuv (or mutant) protein expression using the
auto-induction method
4. Harvest cells and fractionate into soluble and insoluble
fractions
5. Detect GFPuv (or mutant) by SDS-PAGE, Western
blotting and probing with HisprobeTM-HRP
Preparation of competent
cells
Auto-induction
Cell fractionation
SDS-PAGE
Western blotting
Protein expression
lecture (prokaryotic)
Purify GFPuv
protein and analyse
its properties
(practicals10-11)
1. Purify GFPuv (or mutant) using Ni-NTA chromatography
2. Determine the concentration of protein by Bradford
Assay
3. Analyse GFPuv (or mutant) by mass spectrometry &
fluorimetry
Ni-NTA chromatography
Bradford Assay
Mass spectrometry
Fluorimetry
Protein separation
methods:
chromatography
Mass spectrometry &
fluorimetry lectures
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MODULE TIMETABLE
All practical sessions will take place in Laboratory C on level 9 of the Garstang Building, unless
specified otherwise.
Practical Activity
Introduction
27 Sept
Tuesday 10-4pm
10-2pm
x Health & Safety Induction (working in the lab)
x Induction activities including calculations/pipeting
2.30-4pm
x Introductory lecture to practical project and writing lab notes
Practical 1
04 October
Tuesday 10-2pm
x Measure plasmid pET23-gfpuv DNA concentration and purity
x Set up double restriction enzyme digest
x Prepare agarose gels in preparation for Practical 2
Practical 2
11 October
Tuesday 10-4pm
x Analyse restriction digest products by DNA agarose gel electrophoresis
x Extract the gfpuv containing fragment from the agarose gel
x Estimate the recovery of DNA by NanoDrop (UV spectrophotometer)
x Set up ligation of gfpuv fragment into pET28c vector
Practical 3
18 October
Tuesday
10-12pm
x Transform ligated product into E. coli host cell (DH5α cells)
x Finish off literature review and submit by 4pm, 20 October
Practical 4
25 October
Tuesday 9-4pm
9-10am:
x Feedback on GFP literature review (see VLE for venues)
10-4pm:
x Set up colony PCR and analyse PCR products by agarose gel to check
plasmids for insertion of gfpuv
x Design primers for mutagenesis
Practical 5
01 November
Tuesday 10-5pm
x Carry out site directed mutagenesis on gfpuv cloned into pET28c
x Transform products of site-directed mutagenesis into supercompetent
XL-1 cells
Practical 6
08 November
Tuesday 10-2pm
x Extract plasmid DNA from transformation colonies and check purity and
concentration of extracted DNA by NanoDrop (UV spectrophotometer)
x Concentrate the DNA by ethanol precipitation (if necessary)
x Send samples for sequencing
Practical 7
15 November
Tuesday 10-4pm
x Analyse sequencing results
x Prepare competent BL21(DE3) and transform with extracted plasmid
DNA
Practical 8
22 November
Tuesday 10-4pm
x Express GFP protein using the auto-induction method
x Fractionate samples into soluble and insoluble fractions
x Detect protein expression by SDS PAGE followed by Blue Gel stain of
fractionated samples
Practical 9
29 November
Tuesday 10-4pm
x Run SDS-PAGE, electroblot and probe blot with Hisprobe™-HRP.
x Develop blot using a colorimetric assay.
Practical 10
06 December
Tuesday 10-4pm
x Purify his tagged GFP by Ni-NTA chromatography
x Measure protein concentration
x Detect purified GFP by SDS PAGE followed by Blue Gel staining
BIOL5373M Protein Engineering Laboratory Project 2015-16
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YOU MAY COME IN AND COMPLETE LAB NOTEBOOKS DURING THIS
WEEK
CHRISTMAS VACTATION
YOUR PURIFIED SAMPLES WILL BE ANALYSED BY MASS SPECTROMETRY AND
FLURIMETRY AND THE DATA RETURNED TO YOU AFTER THE CHRISTMAS
VACTION
SEMESTER 2
Practical 11
Monday 23
January 2017
This session will be structured as follows:
x 9-10am: End of module test.
x 10-11am: lecture on Fluorimetry and details of assessed coursework
x 11-12noon: Analysis of data
(NOTE: writing research papers and presenting data (graphics) sessions are
scheduled as part of BIOL5271M in the afternoon).
SEMESTER 2
Monday 13
February 2017
4-5pm
Coursework surgery (including feedback on test)
Venue: To be confirm (TBC)
ASSESSMENT and ASSESSMENT DEADLINES
Table 2: Assessment overview
ASSESSMENT
% of formal
assessment
Submission deadline
800 word literature review on “The properties and
uses of GFP”
10% Tuesday 18 October
2016, 4pm
End of module test: this will comprise of a series of
short questions/multiple-choice questions and will
assess the material covered as part of your pre-
practical questions.
25% Monday 23 January
2017, 9-10am
Quality and completeness of information recorded in
the laboratory notebooks
15% Assessed during the
practical sessions
(practicals assessed
will be 5, 7, 9 and 10)
Presentation and critical analysis of results obtained
in the form of a short research paper
50% Thursday 16 February
2017, 4pm.
Total percentage (Assessment Coursework) 100%
Additional information for each assessment including guidelines and marking schemes is
available on the VLE under “module information”.
Work should be handed in to the Graduate School Office, marked for the attention of Dr Chi
Trinh. An identical copy of the work should also be submitted electronically to the assignment
upload area in the BIOL5273M module area on the VLE by the same date.
BIOL5373M Protein Engineering Laboratory Project 2015-16
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COURSEWORK SUBMISSION
All work should be submitted to the Student Education Office marked for the attention of the
module manager and an identical copy uploaded into an assessment upload area of the module
in the Blackboard VLE by the same deadline. A signed Declaration of Academic Integrity Form
should be attached to all assessed work at the time of submission.
INFORMATION ON PLAGIARISM
The University gives clear guidelines on what is deemed plagiarism or fraudulent work and
treats this very seriously. The University has its own website:
http://www.lts.leeds.ac.uk/plagiarism/ which gives advice and guidance to students – please use
it and also read the Masters Bioscience Programme Handbook which also gives details on
plagiarism and cheating.
LABORATORY SAFETY INFORMATION
• All students working in the Garstang Laboratories must familiarise themselves with the
laboratory rules set out below.
• COSHH assessments for individual experiments are on the VLE and should be read prior to
attending the practical.
• You will be provided with a laboratory coat to wear during practical work. Outdoor clothing,
bags, cases etc cannot be taken in to the laboratory. These must be left in the lockers
provided. You will need a 30mm padlock to secure your belongings. This can be
purchased from any supermarket or a hardware store.
General Laboratory Safety Rules
1. Always wear a laboratory coat, properly fastened.
2. Do not smoke, eat, drink or apply cosmetics in the laboratory
3. Suitable eye protection and gloves must be worn in the laboratory when instructed
4. No mouth pipetting is to be carried out
5. Be conscious of hazards. Read the safety notes for each experiment. Report any accidents
however slight to your Demonstrator.
6. Dispose of all broken glass, pasteur pipettes etc into “sharps” containers provided. Never
put them into soft paper waste bins built in under the benches.
7. Most solutions can be safely disposed of down the sinks. Run plenty of water down after
them. But some waste solvents must NOT be put into the sink; pour these into the special
waste containers provided.
8. Keep your area of bench tidy and organized whilst you are working. Mop up spills at once,
first consulting the demonstrator if any hazard is involved.
9. Make sure that you leave your area of the bench CLEAN and TIDY at the end of the
practical.
Mobile phones/ipods/computers/ipads and similar devices should be left in your locker
for the whole practical session.
http://www.lts.leeds.ac.uk/plagiarism/
BIOL5373M Protein Engineering Laboratory Project 2015-16
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Practical 1
The aims of this practical are to:
x Digest plasmid pET23-gfpuv with restriction enzymes NdeI and HindIII.
x Pour agarose gels in preparation for practical 2.
x Measure plasmid pET23-gfpuv DNA concentration and purity.
Experiment 1.1 Digest plasmid pET23-gfpuv with restriction enzymes NdeI and HindIII
Once you have quantified your plasmid DNA, you will “cut out” the gfpuv gene from the pET23
vector by setting up a restriction digest using the two restriction enzymes NdeI and HindIII. To
do this, you will mix 15µl of your plasmid DNA with the restriction enzymes, NdeI and HindIII
and restriction enzyme buffer. The buffer provides the optimal Mg2+, NaCl and pH conditions for
the enzymes to work effectively. Restriction enzymes are extremely temperature-sensitive and
therefore you should ensure that all your components (enzymes, buffer and plasmid DNA) are
kept on ice until you are ready to incubate the reaction mix. The reaction mix will comprise a
total reaction volume of 50µl and will be incubated at a temperature of 37°C for a minimum of 3
hours.
Experiment 1.2 Measuring plasmid DNA concentration and purity using UV
spectrophotometer
You will determine the quantity and quality of the DNA by reading the optical density (OD) of
your sample at 260nm and 280nm (also referred to as A260 and A280).
The reading at 260nm is used to calculate the concentration of nucleic acid in the sample. For
pure, double stranded DNA: 1 OD at A260 = 50µg/ml (for a 1.0cm path length cuvette).
The ratio between the readings at 260nm and 280nm (A260/A280) provides an estimate of the
purity of the nucleic acid. Pure preparations of DNA have an A260/A280 value between1.7-2.0.
Pre-practical preparation
1. Read the protocol carefully and draw a flow diagram to show the main stages of the
experiments.
2. Read through the COSHH form associated with this practical which is available on the VLE.
Background reading:
For a general overview on gene cloning consult:
Divan & Royds (2013) Chapter 1 or Brown, T.A. (2010) – see Reading List
NOTE: there are no pre-practical questions associated with practical 1.
BIOL5373M Protein Engineering Laboratory Project 2015-16
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Experiment 1.1: Setting up a restriction digest
Study Figure 1a for a restriction map of plasmid pET23-gfpuv showing the location of restriction
sites.
In this experiment, you will digest 25Pl of pET23-gfpuv DNA with 40 units of the restriction
enzymes Nde1 and 30 units of the restriction enzyme HindIII in a total reaction volume of 50Pl.
You will need to calculate the volume of restriction enzymes that you will need to add in the
digest below (these are X and Z). The amount of distilled water (Y) that you add can then be
calculated to bring the total volume of the reaction to 50Pl. MAKE SURE YOUR
DEMONSTRATOR CHECKS YOUR CALCULATION BEFORE YOU PROCEED WITH STEP 2.
1. Once you have calculated the volumes, in a sterile microfuge tube, prepare a 50Pl
restriction digest by pipetting together the following in the order shown:
YPl sterile distilled water
5Pl 10x restriction enzyme buffer
25Pl plasmid DNA
XPl NdeI enzyme (20U/Pl)
ZPl HindIII enzyme (10U/Pl)
50 Pl final volume
NOTE 1: Make sure the buffer is properly thawed and well mixed (vortex) before use.
NOTE 2: Add the enzymes last. USE A CLEAN PIPETTE TIP FOR EACH COMPONENT!!
2. After adding the enzymes, mix gently by pipetting up and down (do not vortex) then
close the lid.
3. Incubate the tube at 37oC in a water bath for a minimum of 4 hours.
4. After 4hrs remove the digest from the water bath and store at -20°C until next week. This
will be done for you by the lab technicians.
Experiment 1.2: Preparing agarose gels
You will prepare 1.2% agarose gels in preparation for the electrophoresis you will carry out next
week.
1. Weigh out 1.2g of agarose into a beaker and add 100ml of Tris-acetate buffer (TAE).
The final agarose concentration will be 1.2% (w/v).
2. Place the beaker into the microwave and heat to dissolve the agarose.
3. When the agar has completely dissolved, remove from the microwave and set it aside to
cool to 60°C. Check the temperature with a thermometer.
4. Meanwhile: prepare your agarose gel apparatus by positioning the plastic moulds at
each end. Then position the comb in its designated slot. A demonstrator will show you
how to do this.
5. When the agarose has cooled to 60°C, ask your demonstrator to add 20μl of 5mg/ml Gel
Red (a derivative of ethidium bromide) solution.
BIOL5373M Protein Engineering Laboratory Project 2015-16
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6. Wearing gloves (Gel Red is a safer derivative of ethidium bromide, but still demonstrates
mutagenicity at the concentrations found in the stock solution).
7. Swirl the solution gently to mix, then pour it carefully into the gel mould to a depth of 6-
7mm (usually 30ml) without splashing.
8. Allow the gel to set for at least 30min.
9. These will be stored at 4°C and returned to you next week.
Experiment 1.3 Measuring the concentration and purity of plasmid pET23-gfpuv DNA
solution using UV spectrophotometry
1. Ensure that the spectrophotometer is switched on and the UV lamp is selected. The UV
lamp needs to warm up for at least 10 minutes in order to give an accurate reading.
Check the spectrophotometer is set for 260nm.
2. Take a disposable cuvette being careful to hold it by the matt sides, not the clear sides
through which the light beam will pass. If the clear sides are dirty, clean them carefully
with a little ethanol on a tissue.
3. Carefully pipette 1ml of distilled water into the cuvette and place into the cuvette holder in
the spectrophotometer. Be sure to place the cuvette the right way around.
4. Adjust the reading to zero. This is your blank. Remove it and empty the cuvette
5. Make a dilution of your plasmid solution. As a starting point try 10Pl in 990Pl of water (i.e.
1 in 100). Cover the top of the cuvette with parafilm and invert a couple of times to mix
thoroughly. Remove the parafilm, wipe and moisture from the outside of the cuvette and
place in the spectrophotometer. RECORD THE READING in your lab book e.g.
A260 of 1:100 dilution of plasmid pET23gfpuv=______
6. Carefully transfer your diluted plasmid to a clean microcentrifuge tube and rinse out the
cuvette with distilled water.
7. Set the spectrophotometer to 280nm. Pipette 1ml of distilled water into the cuvette and
zero as you did in step 4.
8. Remove the blank and put your sample back in the cuvette. Measure and record the
reading at 280nm as you did in step 5.
9. Calculate your DNA concentration and the purity of your DNA sample. Record this
information in your laboratory notebook.
BIOL5373M Protein Engineering Laboratory Project 2015-16
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Practical 2
The aims of this practical are to:
x Analyse the restriction digest products (from last week) by agarose gel electrophoresis.
x Extract the gfpuv fragment (761bp) from the agarose gel.
x Check the purity and concentration of the extracted gfpuv DNA by nanodrop.
x Ligate purified gfpuv insert into pET28c expression vector.
Introduction
Experiment 2.1: Analysing your restriction digest by agarose gel electrophoresis
Agarose gel electrophoresis is a common method by which the products of a restriction digest
can be analysed. In this method, an electric current is used to move the negatively charged
DNA molecules across a solid agarose matrix towards the positive electrode. Linear DNA
fragments are separated according to size so that the smaller molecules move faster through
the matrix and therefore migrate further through the gel. Size is not the only factor which
determines the mobility of DNA. The rate of migration is also affected by DNA conformation so
that supercoiled, open coiled and linear fragments (of the same size) migrate at different rates
through the gel.
In this experiment, you will prepare a 1.2% agarose gel and then using a Gilson pipette you will
load your DNA restriction digest samples into adjacent wells of the agarose gel in a horizontal
gel apparatus. Your samples will be loaded alongside a 1kb DNA size marker (also called a
mass ladder).
Before loading your DNA samples into the wells you will add a loading buffer containing the dye,
bromophenol blue. In a 1.2% agarose gel, this dye migrates through the gel at roughly the
same rate as double-stranded DNA fragments of 500bp and is used to monitor visually how far
the DNA has moved through the gel. After electrophoresis, your separated DNA fragments will
be visualised by placing the gel under UV light. We will be using the staining dye, Gel Red,
added to the agarose gel before electrophoresis, to visualise the DNA.
A photograph of how the marker bands will appear on an agarose gel, viewed under UV light is
reproduced below.
Remember: A DNA ladder consists of a series of known DNA fragments of defined sizes that
BIOL5373M Protein Engineering Laboratory Project 2015-16
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are often produced by restriction digest of a plasmid. When run on a gel, the fragments
separate out according to size and appear as a ladder when visualised by ethidium-bromide (or
some alternate method) of staining.
Experiment 2.2: Extracting the gfpuv band from the agarose gel
Once you have identified which is your gfpuv band on the agarose gel: under a UV
transilluminator, you will cut out the gfpuv band from the gel using a scalpel blade. Then you will
go on to purify the DNA from the contaminating agarose and Gel Red stain using the QIAquick
gel extraction kit from Qiagen. The principle of this DNA extraction method is given in the
QIAquick spin handbook (pages 11-13) available on the VLE which you should read before you
come to the practical session.
NOTE: You will require a photograph of the 1kb DNA ladder for use during this practical. This is
available on the VLE. Please download and bring with you to the practical session.
Experiment 2.3: Check the purity and concentration of the extracted gfpuv DNA by
nanodrop.
For more information you should read the information available on the webpage here,
http://www.nanodrop.com/Library/T042-NanoDrop-Spectrophotometers-Nucleic-Acid-Purity-
Ratios.pdf
Experiment 2.4: Ligating gfpuv insert into the pET28c expression vector
In this experiment, you will ligate your purified gfpuv fragment with linearised pET28c plasmid
DNA to form a recombinant DNA molecule, using the enzyme T4 DNA ligase. Both the
linearised vector, pET28c and your gfpuv fragment have been restricted (cut) with the same
restriction enzymes, NdeI and HindIII and therefore have compatible cohesive ends that can
join together to form a circular recombinant DNA molecule. This is an example of directional
cloning as the two ends of the molecules should only be able to ligate into the vector in the
correct orientation.
In a ligation reaction, different ligation products are likely. These could include vector-vector and
insert-insert ligation products as well as the desired vector-insert recombinant ligation product.
When setting up a ligation reaction the goal is to increase the frequency of producing a
recombinant DNA molecule and minimise the frequency of vector-vector and insert-insert
ligation products. Insert to vector ratio, the total concentration of DNA molecules and the purity
of the DNA preparation are all important factors which determine the success of a ligation
reaction producing the desired circular, recombinant DNA molecule.
In your ligation reaction, you will use 8:1 molar ratio of insert to vector. This is calculated as
follows:
Your plasmid pET28c vector is 5.4 kb in size and the gfpuv insert is 0.7kb. Therefore the vector
is approximately 8 times bigger than insert (5.4/0.7= 7.7). You will therefore, take equal
masses of vector and insert, which will give an 8 fold molar excess of insert.
You will be supplied with 50ng of purified pET28c (pre-digested wi
