2011 101 lab 2 tutor template.pdf
TRANSCRIPT
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BIOSCI.101 Laboratory Course
TUTOR:
DEMONSTRATORS:
This Course has six labs:
1. Enzymology
2. Gene Expression
.4. Evolution
5. Blood Glucose
6. Photosynthesis
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Reminder Lab Organisation
Have you handed in your pre-lab assignment? Things to bring to every laboratory:
Manual (having read the lab!)
Pen, marker pens and pencil
Ruler
Calculator
a ora ory oa
If you miss a lab fill out an Absence fromLaboratory form, attach a medical certificate, handit to the Course Coordinator - Mandy Harper.
Your assignment Sheet! Answer as you go & write in pen. Answers are almost alwa s somewhere in the
text in lab &/or lecture guide OR in thepresentation!
PAY ATTENTION
Write carefully and tidily.
We cant mark what we cant read.
Get your assignment sheet SIGNED OFFbefore you leave
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Exercise 2.1 (pg 2.5): RNA hydrolysis
Start with this
exercise
o n ac con ons se u e w
black screw cap)
Acid hydrolysis breaks phosphodiester linkage
Separate nucleotides by chromatography
Laboratory is in two parts
.
2. Control of Gene expression
Read each shaded box in the lab
each experiment
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The Central Dogma of molecular biology
Basically once information is converted to protein, it cannotflow back to nucleic acid
Transcription
DNA
mRNA
In the cell
information is
stored
where?
Copyright 2002 Pearson Education, Inc., publishing as Benjamin Cummings
Translation
ribosome
polypeptide
protein
Nucleic acids store andtransmit hereditary information
ribonucleic acid (RNA) and
deoxyribonucleic acid (DNA).
DNA provides direction for its own
replication.
DNA also directs RNA synthesis and,
through RNA, controls protein synthesis.
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A nucleic acid strand is a polymer of
nucleotides
Each nucleotide consists of three parts
Base + Sugar
=
uc eos e
Another name for a nucleotide is nucleoside monophosphate
There are two types of nucleotides
6-membered ring
6-membered ring
joined to a 5-
membered ring
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DNA base pairing
Base pairing:
A T
G C
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The sugars of one nucleotide are joined to thephosphate of the next nucleotide via
This creates a sugar phosphate backbonePHOSPHODIESTER BONDS
Base pairing:
A T
G C
Nucleic Acid Structure Important Terms
DenaturationDe = Un, Nature = natural state or configuration
GENTLE BREAK WEAK BONDS i.e.
HYDROGEN BONDS BETWEEN BASES
Hydrolysis
From Greek: Hydro = water, Lysis = breakbreaking using water
HARSH WILL BREAK PHOSPHODIESTER
LINKAGES
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Hydrolysis
The covalent bonds connecting monomers in a polymerare disassembled by hydrolysis
In hydrolysis as the covalent bond is broken ahydrogen atom and hydroxyl group from a split watermolecule attaches where the covalent bond used to be
DENATURATION
HYDROLYSIS
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Inheritance is based on replication of theDNA double helix
DNA is relatively stable, hence ANCIENT DNA
DNA double stranded double helix
A sing e stran e extra O group in
presence of OH radicals) can attack thephosphodiester bond
- Spontaneously at neutral pH and is called auto-catalytic-cleavage
Compared to DNA, RNA is relatively unstable
RNA single stranded & extra OH group
OH group can attack the phosphodiesterbond occurs spontaneously at neutral andalkaline H auto-catal tic-cleava e
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Precipitation and denaturation of DNA/RNA
RNA and DNA have different propertiesue o s zes an s ng e ou e-s ran e
nature. To investigate this:
2.2 Add ethanol and NaCl to DNA/RNAand observed any precipitate that forms
2.3 Place DNA and RNA in boiling waterbath for ~5 minutes and repeat
Back to the RNA hydrolysis
The covalent bonds connecting monomers in a polymerare disassembled by hydrolysis
In hydrolysis as the covalent bond is broken ahydrogen atom and hydroxyl group from a split watermolecule attaches where the covalent bond used tobe
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Chromatography and precipitations
Start precipitations exercises 2.2 and 2.3
Hydrolysis cool for 10 minutes whileyou wait load the standards on your plates then spot the plates with the Tube Asamples
ALL DONE? Pick up bacterial plates andstart reading ahead, there will be a lacoperon talk
2.1 RNA hydrolysis
phosphodiester bonds
broken
RESULT: ribonucleotides
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RNA hydrolysed to ribonucleotides
purines nucleotidesunstable, breakdown to
release bases
Four RNA hydrolysis products
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RNA hydrolysis (2.1)
After hydrolysis cool for 10 minutesw e you wa oa e s an ar s on yourplates then spot the plates with theTube A samples
Thin-layer chromatography
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RNA hydrolysis (2.1)
Afer the plates have dried, place in tanks(2 rou s/tank) under su ervisionDevelop for as long as possible (> 1 hour),i.e. visualise ~4:30pm
Thin-Layer Chromatography
standards
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RNA hydrolysis (2.1)
View in UV viewing box (basesa sor uoresce un er g
Thin-layer chromatography
solvent will carry
different molecules
at different rate
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Thin-layer chromatography
Chromatography tanks
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Viewing Chromatograms
Lab 2 Gene ExpressionThe Lac Operon
.
genotype of stains A, B, C
2.5 Induce -galactosidase with
lactose/IPTG & determine effect of
glucose
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B Plate
using this format.
A
B+I Plate
using this format.B
A
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lac Operon in E. coli
repressorpromoter
negative gene regulation
positive gene regulation
via cAMP receptor protein
Low glucose
mRNA
lacZ lacY lacA
mRNAcAMP
permease transacetylase
repressor
protein
lactose
(inducer)
Inactive repressor
+
lactose
-gal = enzyme
lactosegalactose glucose
Transcription of -gal
occurs:Lactose (inducer) is
present
And glucose is absent
(cAMP is high)
lac Operon in E. coli
repressorpromoter
lacZ lacY lacA
mRNAmRNA
permease transacetylase
repressor
protein
IPTG(inducer)
Inactive repressor
+
IPTG
-gal = enzyme
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2.4 Different strains of E.coli
Analysis of plates Think: in which plate is inducer present? B+I plate, no inducer on B plate
Think: what do mutations do? -galactosidase mutation:
non-functional enzyme
Operator mutation: repressor cannot bind i.e. cant switch off
What colour will strains be? - ype
white without inducer, blue with inducer
-galactosidase mutation always white
Operator mutation always blue
2.5 Induce -galactosidase
. ,to tubes as given in book (step 2)
Incubate for 20 minutes at 37C, add lysismedium & tap
Incubate for 15 minutes at 37C
Add ONPG and incubate for 5 minutes
Observe colour of each tube, note colour insome tubes may be pale
Start ASAP, can do other tasks while waitingfor incubations
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XXXX MASTER SLIDE
using this format.