Benzocaine report feedback

1. Catch-up labs,info for Tutors Damien Hardy
Studentsonthe variouscourseshave beenaskedtowrite upthe followinglabs(chosenbytheir
CLs).
BMS: Immunologyand Proteins&Metabolism
BioChem: Any2 from Structural Biology,Cell Biology,PCR MasterclassorBiological Chem
Chem:targetsynthesisproject
Biol: Any2 fromOptimisation, Cell Biology, WesternBlotting,PCRmasterclass
Human Biol: Cell Biology plusone from PCRmasterclass,Optimisation,WesternBlotting
The studentshave submittedviaGrade Centre tothe PSP3LONG Bb site. Please addcommentsto
the scriptsand/or the feedbackbox ongradecentre. Ithinkyoumayhave to add a mark for the
studentstosee the feedback –please put‘1’,to indicate you’ve lookedatit. I’ll tell the studentsit
isn’tan indicationof theirachievement.
Belowisthe template providedforstudents writingup all labs(exceptChemistryproject).Feedback
shouldbe focusedonbeingof use whenwritingupresearchproject. We are not providingamark.
Commentsonclarity,writingstyle,how dataispresented,appropriate figure legendsetcis
appropriate. I’ve managedtogetsome guidance fromcolleagues (seebelow) onwhatthe students
were doingforthe highlightedpracticals.
PSPS3 LAB SUMMARYFORM:
(Note thatthe boxesmaybe expandedwhere appropriate.)
Title (max 30 words)
Your title needs abitmore qualificatione.g.Synthesisof Benzocaine inthe Laboratory

2. Study Aims and Hypotheses (max 200 words):
Methodsincludingdataanalysisandorstatistical analysismethodswhere appropriate (instyle of a
paper) (300-500 words)
Good introduction/background.Collectmore references. Insteadof puttingarrowsondisgramof
benzocaine,use chemdraw andnumberthe carbonatom as shownbelow
Write the (yieldingm,..%) afterthe name of compoundinthe experimental section. The TLCsolvent
usedisincorrect.
0.75g of Pd/Cwasmixedwithethyl 4-nitrobenzoate (4.84g).Mixture wasthenaddedtoml of
methanol.Mixture wasthenaddedto5.17g of ammoniumformate.Reactionmixture wasrefluxed
for 1 hour. Reactionwascooledbyan ice bath.It was filteredthroughcelite.Thenevaporatedusing
a rotary evaporatorto produce benzocaine asasolid.The yieldproducedwas0.46g.1H NMR
(400MHz, d-CDCl3) δ 7.79 (d,1H, J=8.6Hz), 6.57 (d,1H, J=8.7Hz), 4.27 (q,1H, J=7.2Hz), 3.99 (s,1H),
1.30 (t, 1H, J=7.2Hz). You onlyput 1H NMR (400MHz, d-CDCl3) δ it at the beginning
To a mixture of ethyl 4-nitrobenzoate (4.84g,??mol) andPd/C(0.75g) inmethanol (??ml) wasadded
ammoniumformate (5.17g,?? mol) etc

3. Results(only):Include detailedfigureswithcomprehensivelegends,and a brief interpretation of the
studiesfindingandhowtheycorrelate tothe initial studyhypothesis(300-500 wordsexcluding
figuresandlegends)
Damien
Quite good report except that you have errors I have pointed out.
I would number the sub-headings:
1. Introduction
2. Results and Discussion
3. Conclusion
4. Experimental
5. 5.References
Dr Akram Khan
MAKhan
06/03/2022
You needtolabel usingchemdrawyourstructure of benzocaine anduse these numberswhen
interpretingthe HNMRandCNMR spectra.Referto numberedatomsinyourMethodsNMR
description.
I notice the IR spectrumshowstwo>C=O values 1679 and 1633 cm-1
??

4. Conclusion:A summaryof the keyfindingsfromyourdataanalysis(results)(Maximum200words)
Proteins& Metabolismtutor guidance
NickP has providedthe followinginformationfortutors.
Kathy
The report as it stands needsmore informationinsertingin it.It is shallowin introduction.Attend
to all the pointsI have mentionedhere and in your report.
Dr Akram Khan
06/03/2022
You needtobringthe TLC evidence aswell as spectroscopic evidence when discussing the purity
of your benzocaine made in the lab.

5. Proteins& Metabolism
NickP has giventhe followinginfo forthe P&Mlab.
Aims ofthe lab: to isolate,thenpurifythe enzyme alcoholdehydrogenase (ADH) fromyeast.
Studentsundertake isolationbyphysical disruption,thenaseriesof purificationsteps(PEGand
affinitycolumn) generatingsamplesthroughthe isolationandpurificationprocess.Studentsthen
performa seriesof assaystomeasure ADH activityinthese samples.
Methodsused:studentsperformADHisolationwithglassbeads,PEGtreatmentandrunsamples
througha Sepharose columntoelute the enzyme.Thisgenerates3samples – crude extract,
supernatant(afterPEG) andelution(fromcolumn).Studentsassaythese3samplesusinga
spectrophotometrickineticassay(usingan ethanol substrate andmonitoringNAD+toNADH
conversionat340nm). StudentsalsoassessproteinconcentrationusingBCA andare expectedtouse
these valuestoconsiderthe yield(howmuchactivity) andpurity(yieldof enzyme vslossof
contaminatingmaterial).Studentsare providedatable andequationenablingthemtocalculate
activityinstandardunits(Katals) andtotake intoaccount activityandproteinconcentrationto
quantifyyieldandpurity.StudentsthenrunSDS-PAGE,anduse these toa) staintotal proteinwhich
complementstheirBCA assay,b) performanin-gel ADHassayusinga substrate,c) westernblotto
identifyADHthroughantibodybinding.
Results:studentsshouldhave loggedADHactivity(calculatedasKatals) andproteinconcentration
(readfroma standardcurve) ina table whichisthenusedto calculate “yield”and“purification
factor”, anddata presentationwouldinvolve standardmethodsof displayingquantitativedatasuch
as tablesor bar charts. Statistical analysisisnotrequiredasstudentsonlyhave 1readingpersample.
Studentscollectimagesof gels,whichwouldbe presentedasgelscontainingbands(proteinorADH)
alongwitha molecularweightladder.
Discussion:there isa lotof differentassaystodiscuss,with complementarydata.Proteinassays
provide informationonhowpurificationremovesunwantedmaterial (purity) andthe ADH
spectrophotometricassayquantifiesactivity(yield) asthe purificationprocessproceeds - whichcan
be alignedwiththe in-gel ADHassay.Thisin-gelassaysallowsdeterminationof molecularweight(as
confirmatoryevidence thatthe productisthe expectedone),andthisisfurtherconfirmedby
Westernblot.Studentsnormallyobserveagoodyieldinsample 1,frequentlyagoodyieldin sample
2, but significantlossof activitybysample 3,sowouldbe able todiscussthe trade-off between
purityand yield.
Studentshave notbeenaskedfora discussion –theyshouldpull outthe keyfindingsinthe
conclusion. Nick’sinfoabove givesideaswhatthese mightbe.

6. Structural Biology
DavidS has providedthe followinginfo.
They have collected andif they wenttothe post laband paidattention will have fittedFTIRspectra
fromwhichthey can work outthe secondary structural contentof the protein underarange of
conditions. They were givenaspreadsheetthatgenerates simulateddatathatoverlays the
experimental datawithpeakfitting, sothey should be presentingthat.
They will alsohave lambdamax fromTryptophan fluorescence whichisredshifted asthe protein
unfolded. They maypresentthisasspectraor as extracteddata.
The also have thioflavin-tdatathat showsthe protein isamyloid, againthisisafluorescence
emission spectra.
Finally there issome 1DNMR spectraof the protein thatchanges as the protein unfolds. Thisshould
show a lack of spectral dispersion andwill be presented inwrittenform.
So
FTIR – Fittedspectra
Trp fluorescence, spectraorlmax extracted
ThT fluorescence, spectraorlmax extracted
NMR writtendescription.

7. Cell Biologyfrom Neil C
PSPS3 LAB SUMMARYFORM:
(Note thatthe boxesmaybe expandedwhere appropriate.)
Title (max 30 words)
Study Aims and Hypotheses (max 200 words):
Methodsincludingdataanalysisandorstatistical analysismethodswhere appropriate (instyle of a
paper) (300-500 words)
Effectof FCS on proliferationof cancercellsbytwomethods(orwordsto that effect)
Hypothesis:FCSwill enhance proliferation
MTT and Crystal violetwillbothgive similarmethodsastheyare both methodsof assessing
proliferation
Some mightsaythat MTT andCV methodsmeasure differentthings,sohypothesisethattheywill be
different.That’sfine andgood.
Aims:assessproliferationof arange of FCS concentrationsbyMTT and CV methods.
Expectbasiccell culture method,explanationof platingcells,ideallywithcalculationof cell counts
usinghaemocytometer
Expectexplanationof additionof FCS(changingmedium) after3 or 4 daysand thenassessmentof
MTT and CV 3 or 4 days afterthat
Crystal violetmethodexpected
Remove medium,fix inmethanol,dry,addCV solution(itwasreallyweak,soitdidn’tworkwell for
most),wash3x withwater,measure at540ish nm?
MTT methodexpected
AddMTT, incubate 4 hours,remove medium, addisopropanol,measure at570ish

8. Results(only):Include detailedfigureswithcomprehensivelegends,and a brief interpretation of the
studiesfindingandhowtheycorrelate tothe initial studyhypothesis(300-500 wordsexcluding
figuresandlegends)
ExpectMTT and CV data presentedashistograms
Title:Effectof FCSon proliferationof PC3cellsasdeterminedbyMTT (orCV) assay
Graph: Mean +/- SD, withsignificance starswhichlookplausible.Mystockdata has *** for all FCS,
theirdata mightnot
If FCS of 10 and 20% goesdown,that’sexpected(cellsovergrow,orrunout of glucose whichesp.
affectsMTT (succinate dehydrogenase-dependantassay)
Legend:shouldstate itsmean+/- SD, n=12 wells,4daysstimulationswithFCS,andstate whattests
done.IdeallyANOVA orKruskal-Wallace,will acceptT-Tests.
Axis:Abs570 or whatever
X axis:FCS(%v/v).Lose marksformissingorduplicateddetail
If data lookstoo perfectitismy data: theyshouldstate inlegendif itsstandarddata.
Above issomethinglike whatIexpectbutwithlegendbelow.

9. Conclusion:A summaryof the keyfindingsfromyourdataanalysis(results)(Maximum200words)
Biological Chemistry
Some background fromDanny
It’sessentiallythe same reaction(reductionof aketone toa secondaryalcohol) done twodifferent
ways.
In the firstweek, theydothe reduction“chemically”usingsodiumborohydride. Theyalsosetupa
reactionto dothe transformation“biologically”(enzymatically) usingbaker’syeast.
FCS makescell grow:why?ContainsgrowthfactorsleadstoprogressionfromG1-Sphase. Some
may explainthatoptimumdose is10%as use incell culture,otherswill commentondipinabs at
10 or 20% withone or bothassays,whichis alsoto be expected.
Commentif one assaydoesone thingand otherdoesanotherthing,relate backtoCV is stained
adherentcells,MTTis metabolicactivity.Bothare proxymeasuresof actual cell numbers.
Good answerswill linktothe ki67(markerof proliferationthattheydidanimmunofor) which
increasesin+FCSvs -FCS.
Some will mentionflowcytometrydemodatawhere +GCSleadstoincreasedSphase and M
phase and reducedG1.

10. PCR Masterclass from Neil C
PSPS3 LAB SUMMARYFORM:
(Note thatthe boxesmaybe expandedwhere appropriate.)
Title (max 30 words)
Study Aims and Hypotheses (max 200 words):
Methodsincludingdataanalysisandorstatistical analysismethodswhere appropriate (instyle of a
paper) (300-500 words)
Effectof varyingannealingtemperature andMg onoptimisationof PCRspecificity
Aims:varyannealingtemptoidentifyoptimumannealingtemp
Varymg concentrationtoobtainmax specificitywhilstmaintaininggoodamplification
Hypothesis:Highmgwill resultinmore productbutwithreducedspecificity
Hightempwithincrease specificitybutmightreduce amountof product.Low tempwill resultinnon-
specificamplification.Tempabove Tmof primerswouldpreventamplification(buttheydon’tknow
the Tm of the primerssomightnot mentionthis)
Expectfull explanationof PCRsetup:OptimisedPCR(withapremade mastermix)
Setup of varyingMg (*so explainwhatdoesinasingle reactionideallyandstate range of Mg used)
Varyingtemp:Give componentsof whatgoesintoeachreaction(all same here) butstate varying
tempan a gradientblock
Cyclingparametersshouldbe includedforall 3runs
Eg 3min 94, the 35 cyclesof 94 1min,50-65C for 1 min,72 for1 min
Expectmethodsforagarose gel and alsothe polyacrylamidegel (unlessitstatestheywere pre-cast
whichispossible)

12. Results(only):Include detailedfigureswithcomprehensivelegends,and a brief interpretation of the
studiesfindingandhowtheycorrelate tothe initial studyhypothesis(300-500 wordsexcluding
figuresandlegends)
Expect3 gelsall labellednicely
Ideally:Lanesnotlabelled1-8butlabelledwithMgconc or tempor dilution(neat/1/10,water
blank)
All gelsshouldhave mainbandsof ladderlabelled
Pre-optimisedagarose gel (thisimage isfromstandarddata:I don’tlike the lanes1-5being
numbers,butmarkersare labelledwhichsgood).I’dputan arrow on the correct band with224bp
nextto it.See bottomgel.
Theywere giventhisstockdata:They shoulddosome workat presentingitsensiblytomyversion
(bottom)
I’dpresentitlike this
Expecta legendoutliningwhatwasdone andbrief outlineof interpretation.
Note:anyRT-PCR data write upisthemgettingconfused.The technicianssupplieduswithstock
data for the MSC lab incsome real-time PCRdatathat theydidn’tactuallydo.Anyone talkingabout
qPCRor realtime PCRiswingingit!

13. Conclusion:A summaryof the keyfindingsfromyourdataanalysis(results)(Maximum200words)
Biological Chemistry– notesfrom Danny
Relate toaim: optimisationof PCR.Mainvariablesare annealingtemp.Needstobe 2C below
meltingtemp(TM) of primers.ThisisgovernedbyGC content(higherGC,higherTm, higher
annealing).If annealingabove Tmyougetno amplificationasprimerscan’tbind.Verylow
annealing,primersbindnon-specificallyhence the largerbandsat4-500 bp
Mg: affectsfidelityandactivityof DNA pol:toohigh,gethighactivity, low fidelityandnon-specific
bands.Too low,noamplification.Datahere showsoptimal at0.5mM, withnon-specamplification
above this.
Good answerswouldsuggestgoingbackandre-doingtempoptimisationat0.5mM(as they
probablydid1,.5mM) to furtherincrease optimisation.Othergoodanswersmightsaythatthe
primerconcs couldbe titratedas the largerbandcould be asymmetricamplificationpreferentially
form1 primers.Ireallydon’texpectanyone togetthese last2 points,butif theydo,bonus!

14. Biological Chemistry– notesfrom Danny
It’sessentiallythe same reaction(reductionof aketone toa secondaryalcohol) done twodifferent
ways.
In the firstweek,theydothe reduction“chemically”usingsodiumborohydride. Theyalsosetupa
reactionto dothe transformation“biologically”(enzymatically) usingbaker’syeast.
In the secondweek,theyworkupandpurifythe biological reaction.
The product will be the “same”fromboth reactions,butthe productof the borohydride reactionwill
be racemic,whereasthe productof the biological reductionwill be enantio-enriched.Sothe IRand
NMR spectra (assumingtheytookthem) will lookidentical asthese techniqueshave nowayof
differentiatingenantiomers.Othertechniqueslike massspec(whichtheycertainlydidn’tdo)
wouldn’tbe able totell themaparteither.Sointhe lab,theymeasure the optical rotationof both
products.Since chiral moleculesrotate plane-polarisedlight,the racemicsample hasanoptical
rotationof zero,whereasthe enantio-enrichedsample givesarotationvalue (Ican’trememberwhat
it isoff the top of my head.I thinkit’sthe + enantiomerwhichisinexcess).
Optical rotationonlytellsyouwhetheryourmajorenantiomerrotatesplane-polarisedlight
positively(clockwise)ornegatively(anticlockwise)andtowhat degree.If yoursample isn’tchirally
pure (theyneverare),thenit’sthe average of the mixture.Youcan’treallytell chiral purityfrom
optical rotation – you wouldneedchiral HPLCor GC for this.Youcan have a stab,assumingyou
knowwhatthe rotationof the pure enantiomeris,butit’sprettyinaccurate.Youalsocan’t tell
absolute configuration(RorS) fromoptical rotation.The onlyreasonwe can dothis inthiscase is
that someone inthe pasthas assignedthe configurationof the chirallypure product(usinga
technique likeX-Raydiffractionorchiral shiftNMR) andthenmeasuredthe optical rotation,soit
linksone piece of data(R/Sconfiguration,XRD) tothe other(+/– optical rotation).Ithinkinthiscase
the major enantiomerproducedis(S) andthathas a positive rotation,butthisisn’tarule across all
molecules,sosome moleculeshave R:+,S: – and vice versa.
So I guessthe writeupwill be astandardsyntheticlab –two syntheticproceduresdescribingthe
setup,monitoring,workup,purificationof eachreaction.Introdiscussingimportance of chiralityin
biologyandchemistry.R&Ddiscussingwhattheydidandthenmethodsformeasuringchiralityetc
and implicationsforsynthesisof moleculesthatinteractwithbiological systemslikedrugs,
fragrancesetc?

15. Chemistrytarget synthesisproject – from Simon
Level 6 Chemistry catch-up labs portfolio template
Introduction
Figure 1 Suzuki coupling
The diagram Figure 1 shows my reaction new carbon-carbon bond is formed using a
palladium cross coupling. The bond is meta to the pyridine nitrogen on the pyridine rings
and the substituent X is meta to the new carbon-carbon bond.
Molecule identity
Discussion of 1
H and 13
C NMR, MS, high res MS, IR with reference to the structure of the
molecule ideally with a diagram.
Molecule purity
Discussion of 1H NMR centred a round purity of the molecule.
Moving forwards
Something new focusing on structures, mechanisms or literature
Experimental