The aim of the experiment was to isolate a section of human DNA containing the human TATA box binding protein (hTBP) gene using PCR, cut it with restriction enzymes, and insert it into a plasmid vector. The recombinant plasmid would then be transformed into bacterial cells. Gel electrophoresis and qPCR were used to analyze samples and confirm insertion and expression of the hTBP gene. Results showed that purification of the plasmid reduced contaminants, gel electrophoresis indicated restriction enzymes cut the DNA as expected, blue/white screening showed potential recombinant colonies, and qPCR revealed the hTBP gene was expressed in test samples but not controls. The experiment supports the hypothesis that the hTBP gene could be successfully isolated, inserted into

1. JM B9024263 FLR Molecularbiology
Title: PCR amplification of human DNA and insertion into a plasmid vector.
Aim:The aim of thisexperimentwastocut a sectionof humanDNA containingthe humanTATA box bindingprotein,
whichhad beenamplifiedviathe polymerase chainreaction,andsplice itintoacut plasmidvector. Before ligation,
boththe insertandthe plasmidwouldbe analysedbymeansof gel electrophoresisalongwithcontrol samplesto
determine whetherithadbeenproperlycut.The insertwouldthenbe ligatedinto the plasmidvectorviaenzymatic
activity.The recombinantplasmidwouldthenbe introduced andtransformed intoaculture of bacteria.The
subsequentsampleswouldthenbe subjectedto blue-whitescreeningtodetermine whetherthe inserted
recombinantplasmidhadinactivatedthe Lac-Zgene,whichcodesforthe β-galactosidase enzyme whichwouldlyse
lactose intoglucose andgalactose.Shouldthe experimentbe asuccess,the sampleslackingthe recombinant
plasmidwouldpresentblue coloniesasthe testincorporatesthe compoundX-gal (whichisstructurallysimilarto
lactose) whichwouldbe lysedbybacteriawiththe active Lac-Zgene toformblue colonies.The bacterialcellswith
the recombinantinsertwouldpresentonlywhite coloniesastheyare unable tobreakdownthe X-gal.
Hypothesis:If we are able tosuccessfullyisolate DNA fromanoutside source (hTBPgene)andligate itintoa
successfullycutplasmidvectorandtransformsaidrecombinantvectorintobacterial culture,thenthe recombinant
plasmidwill be able toreplicate episomallyinthe bacteriaandwill be able toexpressthe genesitcarries,namelythe
hTBP gene.
Results:
Purification of plasmids
Preparedwere two1:5 dilutionsof plasmid,one whichwascrude andone of whichhadbeensubjectedtoa
purificationprocessinwhichthe plasmidwasfilteredthroughaQIAprepcolumntoremove amajorityof the cell
debriswhichwascontainedwithinthe mixture. Inordertotestthe purityof the E. coli samplesused,the samples
were subjected toUV spectrophotometryand absorbance values of the purifiedsamples andthe crude samples
were comparedinorderto determine whetherthe purificationwassuccessful. The absorbance valueswerethen
insertedintoasuitable chartto betterdisplaythe results(Fig.1)
Fig. 1: A table displayingthe resultsof spectrophotometryof boththe crude andpurifiedsamplesattwodifferent
UV wavelengths(260nm& 280nm).
0.867
0.612
0.384
0.289
0
0.2
0.4
0.6
0.8
1
260nm 280nm
Absorbancevalues of the crude vs purified
plasmid at differentwavelengths
Crude Plasmid Purified Plasmid

2. Gel Electrophoresis
Gel electrophoresiswasthenperformedonfive of ourdifferentsamples,the cutplasmid,the uncutplasmid,the cut
PCR product,the uncut PCRproduct andfinallyanegative control whichservedasamarkerto determine whether
noticeable change hadoccurred. Thistestwasperformedinordertoensure boththe plasmidandPCRproduct had
beensuccessfullycut,essentiallygaugingwhetherthe ligationof the PCRproductintothe plasmidwouldbe
successful. Eachof the sampleswere introducedto 2.5µL of 5x loadingbufferinordertoincrease theirdensity,
allowingthemtosinkinthe sample bedsinthe agarose gel. Thistestwouldgenerate quantitative resultsshowing
howthe DNA fragmentshadbeenseparatedbasedontheirsize andbandshadformed at differentdistancesalong
the gel accordingto the size of the molecule, meaningthe cutproductswouldshow noticeablydifferentresultsto
theiruncutcounterpart.Afterthe processhadfinished,the agarose gel wasthenviewedunderUV lightandwas
photographed(Fig.2)
(Fig.2) The photograph taken of theagarosegelin which the electrophoresishad taken placeat 100v. Labelled
abovetheimage are thenamesof each sampleused. A scale wasnotneeded as the results were purely quantitative
and were gathered in order to see a noticeablechangein oursamplescompared to the controlsto validatethe
successof theexperimentso far.
Note:my personalgelelectrophoresisresults were contaminated in theway of mixing of samplesin the agarosegel
wells and showed very little positiveresults thereforeI decided to use thestandard data forthistest as it better
represented theexperimentsgoalsand hypothesis
Blue/White Screening
Two agar plateswere pouredusing80mg/Lampicillintotestforthe potential antibioticresistancethatthe
potentiallyrecombinant-containingsamplescouldshow (The twoplateslabelledL).Afterthe recombinantplasmid

3. had been transformed intothe bacterial cultures(inbothsampleslabelledL),theywere incubatedforaperiodof
time alongside the positivecontrol whichcontainedthe control plasmidpETBlue andanegative control which
containedsterilewater,bothof whichdidnotcontainampicillin.(Eachsample hadtwoagar platesincubated,one
with5µL of sample andone with 50µL of sample).
The purpose of the blue/whitescreeningwastomake sure that the recombinantplasmidhadbeensuccessfully
transformedintothe bacterial culture andthatithad neitherligatedtoitself beforethe introductionof the insert
DNA or onlya single cuthadbeenmade inthe vector.The latterispossible onlybecauseof the closenessof the
bindingsitesof the twoenzymeswe used(EcoRIandEcoRV) whichcouldhave causedanincomplete cut.
Afterthe sampleshadbeenincubatedsuitably,the numberof coloniesoneachplate couldbe countedandtheir
colourrecorded,the resultsof the agar platesare shownin Fig. 3.
Number of colonies counted and their colour
Fig. 3 The numberandcolourof coloniespresentwithineachsample.X-gal hadbeenintroducedintoeachsample’s
agar, whichissimilarthe lactose instructure butwouldformblue coloniesupondigestionmeaningif the LacZgene
was uninterrupted,bluecoloniesshouldform (5-bromo-4-chloro-3-hydroxyindole isoxidisedinto5,5’-dibromo-4,4-
dichloro-indigo,whichisblue).Therefore,we wouldhopetosee blue coloniesinthe positive control samplesonlyas
the potentiallyrecombinantsample shouldhave hadtheirLacZpathwayinterruptedbythe insertDNA.
qPCR
The purpose of the qPCRisto determine whetherthe insertedhumanTATA box bindingproteinisbeingexpressed
by the bacteriaand to quantifythe level of transcriptionincomparisontothe reference gene,inthiscase HK16S
rRNA. Afterthe qPCRwas performedoneachsample twice (totestforbothgenes) andthe thresholdcycles
measured,the resultswere amassedintoatable tocompare results(Fig.4)
The test wasrun withwellsA1andA4 beingemptyinordertoset a baseline negativecontrol,withnoactivityasa
wayto “zero”/calibrate the machine.WellsA2andA5 were run withourtest samplestotestforthe hTBPgene
expressionandHK16S rRNA gene expressionaccordingly.WellsA3andA6 were runwithcontrol sample inorderto
geta baseline expressionvalueforaregularunmodifiedplasmid. WhilstwellsA2andA5 containedourtestsamples
inwhichwe had hopedto see expressionof the hTBPgene.
Well Sample Name Target Name Cт
A1 hTBP Undetermined
A2 Test cDNA hTBP 10.19
A3 Control cDNA hTBP Undetermined
A4 HK 16S rRNA Undetermined
A5 Test cDNA HK 16S rRNA 12.97
A6 Control cDNA HK 16S rRNA 12.40
Fig. 4 Standard resultsof the qPCRexperimentusedtodetermine the CT of each sample. CT values are relative
measures of the concentration of the target in the reaction.

4. Discussion:
Purificationof plasmids
While examiningFig.1itis clearthat at both wavelengths,therewere similarresultsseen.Afterfilteringthe crude
plasmidsample thoughaQIA prepcolumnto remove cell debris,the absorbance fell atbothwavelengths(from
0.867 to 0.384 at 260nm and from0.612 to 0.289 to 280nm) due to the cell debrisnolongerinterferingwiththe
lightpassthrough.Thisresultindicatedstronglythatourplasmidsample washeavilycontaminatedwithundesired
productsbefore filteringandthatafterfiltering,the plasmidsample waspurer,andthe absorbance value better
reflectedthe concentrationof plasmidwithinthe sample.Furtherpurification/filteringcouldhave beenusedto
remove furtherwaste howeveritwasnotnecessary
Gel Electrophoresis:
Whencloselyexamining the electrophoresisresultsin Fig.2,a distinctdifferencecanbe seenbetweenthe uncutand
cut PCR product.The difference comesinthe form of a muchlightermainbandinthe digestsample aswell asfaint
bandspresentlowerdowninthe digestsample.Thisindicatesthatthe desiredDNA presentinthe uncutsample had
beenat leastpartiallyorfullyisolatedfromthe undesiredproducts,whichwasthe intendedresult. However, this
couldpotentiallybe puttodoubtdue tothe mainbandsharingthe same distance alongthe gel withitsuncut
counterpartpotentiallyimplyingthatthe DNA hadnot properlybeenisolated.
In the nexttwowellsinthe agarose gel were the cutand uncut plasmidsampleswhichhadmuchmore stark
differencesintermsof appearance onthe agarose gel underUV.The regularplasmidhada muchwider range of
bandsat varyingdistanceswhereasthe digestsample hadasingle brightbandwhichalsoindicatedasuccessful cut
viathe enzymaticactivity,isolatingthe desiredsectionof the plasmidDNA.Thismethodof electrophoresisisvery
basicand yieldsverylowresolutionandqualityresultsandtherefore leadstounclearreadings,if anotherformof
electrophoresiswasperformedonthe samples,suchaspolyacrylamidegel electrophoresis(PAGE) ahigher
definitionsetof resultscouldbe gatheredallowingclearerreadings.
Blue/White Screening
The resultsof our blue/white screening,depictedinFig.3,representthe numberof colonieswhichgrew and
whetherornot insertionalinactivationof the LacZpathwayhad occurreddue to the insertionof ourrecombinant
plasmidinourL samples.The negativecontrol plate showedacomplete lackof anyculturesof anykindas there was
no bacteriaaddedto thissample.The positive control platesshowedthe completeopposite,havingnumerous
culturesof eachcolour at eachvolume of sample. Thisresultistobe expectedinnormal plasmidsinthe presence of
x-gal,meaningbothblue andwhite colonieswouldform astheywouldbe able tometabolise the x-gal.Inthe L
sample plates,we expectedtosee anumberof white coloniesineachsample butnoblue coloniesasthe LacZ
pathwayshouldbe interrupted.Inthiscase we saw onlya few white coloniesinthe 50µL sample plate witha
complete absence of blue colonieswhichwouldsupportourhypothesisif notforthe verysmall numberof white
coloniesdisplayedineachsample asitcouldbe concludedthatoursample hadtoo little bacterial growthoverall to
be conclusive.However,Ibelievethisresultdoessupportourtheory,if onlyloosely.
qPCR:
The resultsof the qPCR were verytellingandconclusive.InwellsA3andA6 were ourcontrol samples,measuringthe
expressionof boththe hTBPgene and the HK 16S rRNA gene,respectively.The latterof the tworeturnedapositive
resultwitha CT value of 12.40 whereasthe formeryieldednoresult,thiswastobe expectedasthe hTBPgene isnot
presentinthe control sample andwouldnotbe able to expressthe gene tohitthe thresholdlevel togive aresult.
However,inourpotentiallyrecombinantsamplesinwellsA2andA5, the A5 well presentedthe control gene ina
similarnumberof cycles asthe control sample however, the hTBPgene hitthe thresholdinA2after10. 19 cycles

5. implyingthatourrecombinantplasmidhadenteredthe bacterial culturesuccessfullyandisreplicatingepisomally,
expressingandamplifyingthe hTBPgene.
Conclusion:Thisexperimentcouldnotbe completedinpersondue tothe pandemic.Furthertestswouldhave taken
place such as EMSA to testfor hTBP synthesishadthe full three daysbeenused.
However,itisclearto see that the resultsthatwe were able tocollectas well asthose providedshow thatwe were
able to successfullytransplantthe sectionof DNA containingthe hTBPgene intoaplasmidandtherefore transform
it intobacterial culture successfully,withthe insertedrecombinantplasmidable toreplicateepisomallywiththe
abilitytoexpressthe genesitcarriessuccessfully.
Our hypothesiswasfullysupportedbyourresultsaswe clearlyrecordthat our recombinantplasmidwasexpressing
the hTBP gene afterbeinginsertedintobacterial culture.
It mustbe noted thatdueto the restrictions wemustabide by due to the COVID-19pandemic,thethird laboratory
day wascancelled and no studentswereable the carry outthe qPCRexperimentsin person,subsequently,I
personally wasnotable to attend the second lab day either dueto COVIDshielding of a person in my household.
Therefore, the second- and third-days resultshad to be taken fromthestandard data sheetsorfrom colleague’s
results.

6. Faculty of Health and Wellbeing - Department of Biosciences
ASSESSED WORK FEEDBACK FORM
Student Name: Jim Machin
Student ID number:
Module Title: Professional and Scientific practice 2
Title of coursework Laboratory reporting - Extended report
Marker: TN
MARK*:
51.5
Strengths:
Results described and all calculations carried ut correctly.
Figures are numbered with correct figure legends and discussion links to the literature with citations
Suggestions for Improvement:
More detail needed in your results
More depth in your discussion and use citations to back up your arguments
Link you work to other works in the literature
Include a reference list
Student comments for Feed-forward (how will you use this feedback to improve your future work?):

7. SIGNATURE DATE:
*Unratified mark.

8. Indicator
First
(High)
First
Upper Second
Lower Second Third Fail Fail mark
Hypothesis,
Aims and
Objectives
(weightingx
1.5)
Exceptional know ledge
and understanding of
the subject and its
underlying concepts
Hypothesis is relevant
and clearly stated.
Concise and appropriate
aims and objectives for
experiment outlined. All
elements of report
introduced in a correct,
clear, concise manner.
No errors.
Excellent know ledge of the
subject beyond w hat was
taught. Hypothesis is
relevant and clearly
stated. Concise and
appropriate aims and
objectives for experiment
outlined. All elements of
report introduced in a
correct, clear, concise
manner. Very minor
errors.
A very good breadth of
know ledge and
understanding relating
facts and concepts
together. Hypothesis is
relevant and clearly
stated. Concise and
appropriate aims and
objectives for experiment
outlined. All elements of
report introduced in a
scientifically correct
manner. Minor errors.
A good breadth of
know ledge and
understanding. Hypothesis
is stated, but may be
unclear. Aims and
objectives stated, but may
be unclear or limited. All
areas of the report
introduced, but lack of
understanding show nin
some areas.
Know ledge and
understanding is sufficient to
deal w ith terminology, basic
facts and concepts.
Hypothesis is stated, but
may be unclear or
incomplete. Aims and
objectives stated, but may
be unclear, limited or
incomplete. Most areas of
report introduced, may show
lack of understanding.
Insufficient know ledge
and understanding of
the subject and its
underlying concepts.
Hypothesis absent.
Statement of aims
unclear, limited or
incomplete. Introduction
incomplete and contains
major errors in
understanding.
Highly insufficient
or no evidence of
know ledge or
understanding of
the subject. No
statement of aims
or hypothesis.
Introduction
missing, irrelevant
or inaccurate in
the most part.
10
Results
(weighting
x3 )
Data presentation is
exceptional, clear w ell
labeled graphs, images
and annotations clear
legends and correct
statistics provided. Clear
descriptions of data
given. No errors in
description and no
mixing of discussion
points in results
Data presentation is
excellent, clear graphs,
images and annotations
clear legends and correct
statistics provided. Clear
descriptions of data given.
Data presentation is very
good. Clear and
appropriate images and
graphs w here needed.
Figure legends included
and descriptive, and data
described clearly and
statistical analysis
performed w here
appropriate.
Data presentation is good.
Appropriate graphs and
images w here needed.
Figure legends included
and descriptive, and data
described.
Data presentation is
satisfactory. Some attempts
made to present data in
sensible fashion but may be
reparative and inclusion of
raw data, some descriptions
given
Data presentation is
insufficient. Some data
presented but not
accurately given in
graphs and limited
descriptions of data.
Limited results,
some graphs or
raw data given
19.5
Discussion
(weighting
x4)
Content is relevant and
is fully evaluated.
Exceptional know ledge
of theory and concepts,
w ith creative and
imaginative application.
Draw s on a w ide range
of sources w hich are
themselves evaluated.
Links results gained
clearly to published
literature
Links results gained
clearly to published
literature
Relevance of content to
subject of coursew ork is
fully evaluated. Excellent
know ledge of theory and
concepts, w ith relevant
application. Draw s on a
w ide range of sources.
Aims of lab clearly given
and key results provided,
linked to some references.
Relevance of the content
to topic is very w ell
reasoned. Very good of
theory and concepts, w ith
aspects w ell explained
and applied. Draw s on a
w ide range of sources.
Summary of results given
and links to literature
made. Relevance of the
content to topic is
reasoned. Good
know ledge of theory and
concepts, with reasonable
attempt to explain to
demonstrate
understanding. Draw s on
a range of sources
Summary of results given
and some links to literature
made. Relevance of the
content to the topic is
generally described although
may be w eak in places or
lacking depth. Satisfactory
know ledge of theory and
concepts w hich is linked to
the scope of the study.
Relies on main recognized
sources e.g. directed texts.
Summary of results may
be given. Choice of
content w eakly justified,
only descriptive use of
know ledge. Little
indication of relevance
of theory and concepts,
confused application of
the know ledge to topic
limited sources of
information
Limited, summary
of results may be
given. Inaccurate
and irrelevant
content,
know ledge or
theory and
concepts.
Confused
application
know ledge to
problem. Very
limited sources of
16

9. Class CG% General Characteristics L5
FIRST
96
Exceptional knowledge and understanding of the subject and its underlying concepts; critical evaluation/synthesis/analysis and of
reading/research; evidence of breadth and depth of reading/research to inform development of work; exceptional demonstration of
relevant skills; excellent communication; performance in some, if not all, areas deemed beyond expectation of the level.
89
81 Excellent knowledge of thesubjectasthestudent istypically able togobeyond what hasbeentaught (particularly forahigh 1st
); evidence of
breadth of reading/research to inform development of work; excellent demonstration of relevant skills; demonstrates strong
communication skills.
74
UPPER SECOND
68 As below but very good work characterised by evidence of wider understandingof the subjectas the student is typically able to relate
facts/concepts together with some ability to apply to known/taught contexts; identification and selection of material to informdevelopment
of work; very good demonstration of relevantskills;demonstrates good communication skills.
65
62
LOWER SECOND
58 Agood breadth of knowledge and understanding of thetaughtcontent although balanced towards the descriptive rather than analytical; uses
set material to inform development of work; addresses all aspects ofthe given brief; good demonstration of relevant taught skills,
55
information or
inappropriate.
Formatting
and
Referencing
(weighting
x 1.5)
Recent review s and
landmark primary
papers cited.
Appropriate academic
and professional
standard, w ith creativity
in the use of language.
Well-presented data
Refs correct and thorough.
Bibliography complete,
and properly laid out. Very
minor errors.
Appropriate academic and
professionalstandard, with
w ell presented data.
References accurate.
Bibliography complete and
properly laid out. Minor
errors. Generally of an
appropriate academic and
professional standard.
Data are clearly
presented.
May use older review sand
may not use landmark
papers. Generally correct
but needs some attention.
English is clear and
appropriate. Data are
clearly presented.
Citation and referencing is
accurate and related to
references in the text.
Some incorrect referencing
and incomplete or not
properly laid out
bibliography. English is
understandable. Data are
clearly presented.
Citation and referencing is
generally accurate and
related to references in the
text.
Little or no proper
referencing.
Bibliography inadequate.
English may be
confused and
inappropriate. Main data
are poorly presented.
Citation and referencing
is inaccurate and
unrelated to references
in the text.
English is
generally
confused and
inappropriate.
Most data are
poorly presented.
Citation and
referencing is
inaccurate and
unrelated to
references in the
text.
6

10. 52 though may be limited in range; communication shows clarity but structure may lack coherence.
THIRD
48 Knowledgeandunderstanding issufficient todealwithterminology,basicfactsandconceptsbutfailstomakemeaningful synthesis;relies on set
material to informdevelopment of work; generally addresses mostof the requirements of the given brief; adequate demonstration of
relevant skills over a limited range; communication/presentation is generally competent but with some weaknesses.
45
42
FAIL
35
Insufficient knowledge and understanding of the subject and its underlying concepts; some ability to evaluate given reading/research
however work is more generally descriptive; naively follows or may ignore set material in development of work; given brief may be only
tangentially addressed or may ignore key aspects of the brief; demonstration of relevant skills over areduced range; communication shows
limited clarity, poor presentation, structure may not be coherent.
25
15 Highly insufficientor no evidence of knowledge or understandingof the subject; understanding of taught concepts is typically at the word
levelwithfacts beingreproducedin adisjointed or decontextualised manner; ignores setmaterial in developmentof work;failsto address most
or all of the requirements of the brief;failsto demonstrate relevant skills;lacks basic communication skills.
5
ZERO 0 Work of no merit OR absent, work not submitted, penalty in some misconductcases.