This lab session aims to familiarize students with polymerase chain reaction (PCR) by having them amplify different truncated sequences of the human DNA glycosylase NEIL3 gene from plasmids. Students will prepare PCR reactions containing primers specific to each NEIL3 truncation, run the reactions, and analyze the resulting products on an agarose gel to determine the size of each truncation. The experiment uses five plasmids containing truncated versions of increasing length of the NEIL3 cDNA, from 843 base pairs to the full-length 1506 base pairs. Comparing the bands on the gel will reveal the different sizes of the truncations.

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Analytical Methods Lab Class
Introduction
The purpose of this lab session is to give you hands-on
experience of the polymerase
chain reaction (PCR). For this, you will be given five different
DNA plasmids, four
containing different truncated cDNA sequences and the fifth
containing the full-length
cDNA for the human DNA glycosylase NEIL3. You will prepare
the PCR reaction mixes,
run the PCR in a thermocycler and determine the size of the
cDNA by agarose gel
electrophoresis.
Nei-like 3 (or endonuclease VIII like 3) is the largest of a
family of three proteins found in
mammalian cells. Each acts as a DNA glycosylase, releasing
oxidized bases from double-
stranded and single-stranded DNA. However, in addition to the
N-terminal Fpg/Nei DNA
glycosylase domain, NEIL3 also has an extended C-terminal tail
of unknown function
comprising several zinc finger domains (Liu et al., 2013). In
order to study the function of

2. these C-terminal domains, several truncations to the hNEIL3
cDNA have been made in
our lab. The shortest cDNA (843 bp) contains only the DNA
glycosylase domain and has
been shown to have this activity. Subsequent truncated cDNAs
contain more and more of
the zinc finger domains at the C-terminus (1044 bp, 1206 bp,
1506 bp and full-length).
The plasmid used in these experiments (pETDuet2) has been
designed for the expression
of active NEIL3 protein in bacterial (Escherichia coli) cells.
Because the DNA glycosylase
activity of NEIL3 depends on the removal of the N-terminal
methionine residue and the
endogenous E. coli enzyme is not active when the penultimate
amino acid is valine (as for
NEIL3), the plasmid also codes for a mutated version of the E.
coli methionine amino-
peptidase (EcoMapY168A; Liu et al., 2012). Thus, the plasmid
is termed a bicistronic
vector as two proteins are expressed from the same plasmid.
PCR primers are single-stranded oligonucleotides that anneal to
either end of the DNA
sequence to be amplified. Here, the forward primer anneals to
the start of the NEIL3
sequence and the reverse primer delineates each of the four
truncated cDNA sequences
and the full length cDNA. Please note that each of the reverse
primers also contains a
non-template XhoI restriction site, preceded by CCG, to aid
downstream cloning. The
DNA sequences of the primers are given in Table 1.

3. Please follow the instructions carefully to improve your chances
of success!
page - 3 of 7
Table 1. DNA sequences of the hNEIL3 PCR Primers.
Name DNA sequence Tm
hNEIL3 Forward ATG GTG GA A GG A CCA GGC TGT ACT
CTG AAT 73.2°C
843-XhoI reverse CCG CTC GAG TTT TTG ACA GTG AGG
ACA GAA ATA TGT CAT
TCT GT
72.1°C
1044-XhoI reverse CCG CTC GAG TGA ATC AAT AGG CCT
TGA GGT CAA GC 70.7°C
1236-XhoI reverse CCG CTC GAG ATC TAG TAT CTG GTT
TTG CTT TGT TTT TCT
TTC CAA AG

4. 71.9°C
1506-XhoI reverse CCG CTC GAG AGG ATT TAA GGT ACG
AGG GCC ATC TGT 70.4°C
Full-length-XhoI
reverse
CCG CTC GAG GCA TCC AGG AAT AAT TTT TAT TCC
TGG CC 71.9°C
NM_018248.2:118-1935 Homo sapiens nei like DNA
glycosylase 3 (NEIL3), mRNA
ATGGTGGAAGGACCAGGCTGTACTCTGAATGGAGAGAAG
ATTCGCGCGCGGGTGCTCCCGGGCCAGGCGG
TGACCGGCGTGCGGGGAAGCGCTCTGCGGAGTCTGCAGG
GCCGCGCCTTGCGGCTCGCAGCCTCCACGGT
TGTGGTCTCCCCGCAGGCTGCTGCACTGAATAATGATTCC
AGCCAGAATGTCTTGAGCCTGTTTAATGGA
TATGTTTACAGTGGCGTGGAAACTTTGGGGAAGGAGCTCT
TTATGTACTTTGGACCAAAAGCTTTACGGA
TTCATTTCGGAATGAAAGGCTTCATCATGATTAATCCACT
TGAGTATAAATATAAAAATGGAGCTTCTCC
TGTTTTGGAAGTGCAGCTCACCAAAGATTTGATTTGTTTC
TTTGACTCATCAGTAGAACTCAGAAACTCA
ATGGAAAGCCAACAGAGAATAAGAATGATGAAAGAATTA
GATGTATGTTCACCTGAATTTAGTTTCTTGA
GAGCAGAAAGTGAAGTTAAAAAACAGAAAGGCCGGATGC
TAGGTGATGTGCTAATGGATCAGAACGTATT
GCCTGGAGTAGGGAACATCATCAAAAATGAAGCTCTCTTT
GACAGTGGTCTCCACCCAGCTGTTAAAGTT
TGTCAATTAACAGATGAACAGATCCATCACCTCATGAAAA
TGATACGTGATTTCAGCATTCTCTTTTACA
GGTGCCGTAAAGCAGGACTTGCTCTCTCTAAACACTATAA
GGTTTACAAGCGTCCTAATTGTGGTCAGTG

5. CCACTGCAGAATAACTGTGTGCCGCTTTGGGGACAATAAC
AGAATGACATATTTCTGTCCTCACTGTCAA
AAAGAAAATCCTCAACATGTTGACATATGCAAGCTACCG
ACTAGAAATACTATAATCAGTTGGACATCTA
GCAGGGTGGATCATGTTATGGACTCCGTGGCTCGGAAGTC
GGAAGAGCACTGGACCTGTGTGGTGTGTAC
TTTAATCAATAAGCCCTCTTCTAAGGCATGTGATGCTTGC
TTGACCTCAAGGCCTATTGATTCAGTGCTC
AAGAGTGAAGAAAATTCTACTGTCTTTAGCCACTTAATGA
AGTACCCGTGTAATACTTTTGGAAAACCTC
ATACAGAAGTCAAGATCAACAGAAAAACTGCATTTGGAA
CTACAACTCTTGTCTTGACTGATTTTAGCAA
TAAATCCAGTACTTTGGAAAGAAAAACAAAGCAAAACCA
GATACTAGATGAGGAGTTTCAAAACTCTCCT
CCTGCTAGTGTTTGTTTGAATGATATACAGCACCCCTCCA
AGAAGACAACAAACGATATAACTCAACCAT
CCAGCAAAGTAAACATATCACCTACAATCAGTTCAGAATC
TAAATTATTTAGTCCAGCACATAAAAAACC
GAAAACAGCCCAATACTCATCACCAGAGCTTAAAAGCTG
CAACCCTGGATATTCTAACAGTGAACTTCAA
ATTAATATGACAGATGGCCCTCGTACCTTAAATCCTGACA
GCCCTCGCTGCAGTAAACACAACCGCCTCT
GCATTCTCCGAGTTGTGAGGAAGGATGGGGAAAACAAGG
GCAGGCAGTTTTATGCCTGTCCTCTACCTAG
AGAAGCACAATGTGGATTTTTTGAATGGGCAGATTTGTCC
TTCCCATTCTGCAACCATGGCAAGCGTTCC
ACCATGAAAACAGTATTGAAGATTGGACCTAACAATGGA
AAGAATTTTTTTGTGTGTCCTCTTGGGAAGG
AAAAACAATGCAATTTTTTCCAGTGGGCAGAAAATGGGC
CAGGAATAAAAATTATTCCTGGATGCTAA
Figure 1. CDS sequence of hNEIL3. Note the position of the
forward and reverse primers to
amplify the full length CDS sequence (minus the stop codon).

6. page - 4 of 7
Protocol
In this experiment you will use PCR to amplify human NEIL3
(hNEIL3) CDS (coding
sequence) cDNA inserts from a bacterial expression plasmid. As
the hNEIL3 cDNAs are
all a different size you should obtain different sized bands when
the PCR products are
separated by agarose gel electrophoresis. The PCR primers used
to amplify the different
hNEIL3 cDNAs are shown in Table 1. The forward primer is
common to all the PCR
reactions as it anneals to the start of the coding sequence of the
cDNA of hNEIL3. There
are five different reverse primers, each annealing to a different
part of the hNEIL3 CDS.
For the PCR you will use a proprietary mixture of dNTPs, Taq
DNA polymerase and buffer
called ‘MyTaq Red Mix’ (Bioline). This should reduce pipetting
errors and ensure better
consistency of results.

7. PCR reaction Mixture
1. Label five 0.2 mL microcentrifuge tubes (very small) 1 – 5,
carefully to ensure that they
can be clearly identified as yours. Using a new pipette tip for
each reagent, add 5 PL
of the DNA plasmid, to each tube as outlined in Table 2.
Continue to add the reagents
as directed in Table 2, making sure you add the correct reverse
primer to the correct
PCR reaction. Make sure you thoroughly mix the ‘MyTaq Red
Mix’ with the other
reagents in the tube. This can be achieved by pipetting the
mixture up and down, but
make sure you only push the plunger down to the first stop, or
you will get
bubbles in your reaction mix.
x Note: It is good practice to tick the reagent after you have
added it to avoid
adding reagents twice or not at all.
Table 2. PCR Reaction Mixes
Tube 1 Tube 2 Tube 3 Tube 4 Tube 5

8. 0.2 ng/Pl DNA 5 Pl 5 Pl 5 Pl 5 Pl 5 Pl
2 PM Forward
Primer
5 Pl 5 Pl 5 Pl 5 Pl 5 Pl
2 PM Reverse
Primer*
5 Pl
*(843)
5 Pl
*(1044)
5 Pl
*(1236)
5 Pl
*(1506)
5 Pl
*(FL)
dH2O 10 Pl 10 Pl 10 Pl 10 Pl 10 Pl
MyTaq Red Mix 25 Pl 25 Pl 25 Pl 25 Pl 25 Pl

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Once the reaction mixes are complete, take your five tubes to
the PCR machine and a
demonstrator will load them into the block of the PCR machine
(thermocycler).
2. The PCR reaction will take about 60 minutes and consists of
the reaction conditions
shown below:
Initial denaturation 95°C 30 s
Denaturation 95°C 10 s
Annealing 60°C 10 s 30 cycles
DNA synthesis 72°C 60 s
Final DNA synthesis 72°C 120 s
3. Once the reaction is complete, collect your five PCR tubes
from the PCR machine.
Remove 5 Pl of each reaction mix to one of five labelled 0.5 ml
tubes and add 5 Pl of
loading buffer and 10 Pl of dH2O to give a final volume of 20
Pl.
Prepare a 0.8% agarose gel

10. 4. Prepare the agarose gel running buffer by diluting the 10x
stock solution of TBE (Tris-
borate-EDTA) to 0.5x (i.e. a 20 fold dilution). This is most
easily achieved by pouring
475 mL of deionised water into a 500 mL measuring cylinder
and carefully pouring 25
mL of the 10x TBE buffer solution up to the 500 mL mark on
the cylinder. Remember
to mix the diluted buffer solution by stretching Parafilm over
the cylinder and carefully
inverting the cylinder 2 – 3 times while holding the Parafilm in
place with one hand.
5. Weigh out 0.24 g of agarose into a 100 - 150 mL conical
flask and add 30 mL of 0.5x
TBE buffer. Place a piece of cotton wool loosely into the top of
the conical flask.
6. Dissolve the agarose by heating in a microwave oven. Do not
exceed the “Medium”
setting on the microwave. The mixture will need to boil to
dissolve the agarose, but
heat it in small bursts (e.g. 30 s at a time) and swirl the flask to
minimise superheating.
7. Once the agarose has dissolved and there are no solid
particles left, allow the mixture

11. to cool to around 55°C.
page - 6 of 7
8. Add 30 Pl of GelRed (a 1:1,000 dilution) to the liquid
agarose and swirl the flask to
ensure thorough mixing.
9. Pour the gel into the gel tray and insert the comb to create
the wells and leave to set
for about 30 min, while you purify the plasmid DNA.
Prepare samples and load on gel
10. Remove the comb from the agarose gel and place the gel and
tray in the
electrophoresis chamber, with the wells at the negative
electrode (The DNA will
migrate towards the positive electrode in an electric current).
Fill the tank with 0.5x
TBE buffer.

12. 11. In lane 1, add 10 Pl of the DNA size marker, Hyperladder I
(See Figure 2). For the
PCR reactions, mix the 20 Pl samples prepared in step 3 and add
10 Pl to each well.
12. Once the lid is in place, switch on the power and set it to
deliver 100V. Check that
current is flowing through the gel (look for bubbles at the
negative electrode).
Electrophoresis should take 40 to 45 min: STOP the
electrophoresis before the blue
dye runs off the end of the gel!
13. Once the electrophoresis is complete, the gel is placed on a
UV-transilluminator to
visualise the DNA bands (Gel Red is excited [fluoresces] at 250
– 300 nm when bound
to DNA). The jpeg files will then be posted on Blackboard.
14. A lab report containing the cropped and labelled gel picture
should be included
in your lab report.
15. You should also include the CDS sequence of hNEIL3 and
mark the positions of
the five different reverse primers used in the experiment (the
full length reverse
primer is given to you).

13. Figure 2. Migration of DNA fragments in Hyperladder I in a
1% agarose gel. (Bioline)
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ANALYTICAL METHODS PCR WORKSHOP 2016-2017
Instructions and Marking Scheme for your Workshop write-up
Your report should be structured into the following sections;
Title, Abstract, Introduction,
Methods, Results, Discussion and Reference List. Write what
you actually did in the
lab and any problems you encountered. Remember, a good
report will contain evidence of
independent research around the subject and will not just
reproduce the information in the
protocol sheet. A lab report should be around 1500 - 2000 words
in length.
1. Abstract: A short paragraph of about 250 words summarizing
the background, aims,
methods and results of the experimental work. (10 marks)
2. In the Introduction, write a brief overview of the subject
matter. In this case, research
the area of base excision repair and NEIL3 in particular and

14. give a brief overview of PCR.
Include references from PubMed or a similar database. End the
Introduction by indicating
what the report is going to describe, i.e. the objectives. (25
marks)
3. In the Methods Section, write what you actually did in the lab
and any problems that
you encountered in your own words and in the past tense. Under
no circumstances
should you ever just reproduce the text in the Workshop manual
(plagiarism). Write
in complete sentences and paragraphs and do not use bullet
points. (15 marks)
4. In the Results Section, you should describe the results you
obtained. Write in
sentences and in paragraphs. Remember that you must label any
figures you include in
your report. Figures must also have a Figure title that
adequately describes the content of
the Figure and where appropriate a Figure legend that gives
details of, e.g. the different
samples in each lane of an agarose gel. (25 marks)
5. For your Discussion, you should expand on the results you
obtained, basing your
discussion on the published literature. For example, here you
can speculate on the
function of the different conserved domains that have been
deleted in the different
truncated human NEIL3 cDNAs. (15 marks)

15. 6. Reference List: Please use PubMed to find appropriate
scientific articles. All
references should be from peer-reviewed published articles or
reviews. Do not reference
websites unless there is no alternative, e.g. for referencing the
Bioline website for a
product manual. The inclusion of 5 – 10 references would be
acceptable and these are
not included in the overall word limit. (10 marks)
Use the Harvard System for listing references, e.g.
Silva, D. and Kompany, V. (2012) Title. J. Biol. Chem. 298,
2565-2574.
For further information, see ‘Module Information’ on
Blackboard
Remember, a good report at Masters level will contain evidence
of independent
research around the subject and will not just reproduce the
material in the handout.