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(1)PCR experimentKey

(2)Summary TableE. coli strain tested

Test performed A B C D E FAmpicillin

Resistant(R) orSensitive (S)?

R R R S R S

KanamycinResistant(R) or

Sensitive (S)?

S S S R R S

Immunodetectionwith anti-GSTNegative, weak

or strong

Negative Negative Strong Negative Negative Negative

Size of PCRproduct withprimer pair X

900bp N/D N/D N/D N/D N/D

Size of PCRproduct withprimer pair Y

N/D 500bp N/D N/D N/D N/D

FluorescenceMicroscopy

Green

fluorescence ornot

NotGreen

Not green Notgreen

Green N/D N/D

Gene identityfrom

bioinformaticsanalysis

Small GTPBinding

Protein A

Anantibody

N/D N/D N/D N/D

Identity ofcloned gene

product

Small GTPBinding

Protein A

Anantibody

GST GFP KanamycinResistance

None

M

100bp Molecular marker

Ax

Strain A with Primer X PCR

ByStrain B with Primer Y PCR

Cx

Strain C with Primer X PCR

Dy

Strain D with Primer Y PCR

Cx &Dy lanes are meant to controls as C&D strains do not anneal with X or Y primers. Ideally

any bands in the lanes indicate DNA contamination. False positive bands in Cx &Dy lanes due to

mis rimin of X&Y to C&D in PCR

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(3)Further experimentStrain C shows a blue colour when anti-GST antibody is applied. To confirm C is the GST strain, a PCR

reaction should be carried out, followed by DNA sequencing.

Two primers designed specific to the PCR of GST gene are shown in Fig.1. These primers should ideally be

50% G:C content and primers should not be complementary to each other.

In PCR, primers are mixed together with Taq DNA polymerase, dNTP, MgCl2 and the C strain DNA. Below

are the steps for PCR for 30 cycles (From Step 2 Step 4):

1. 95C 5mins (dsDNA to ssDNA denaturation)2. 95C 30sec (dsDNA to ssDNA denaturation)3. 55C 30sec (Primer annealing)4. 75C 2mins (DNA polymerisation)The PCR product and a 100bp molecular maker are then subjected to gel electrophoresis. The major band in

the gel is cut and DNA is purified by denaturing agent such as phenol, followed by ethanol precipitation.

The purified DNA goes through Cycle sequencing, a variant of Sanger sequencing. Di-deoxyribonucleotides,

each labelled with a fluorophore of a different colour, terminates elongation on the template due to a lack of

3OH to form phosphodiester bond with the next nucleotide. The purified DNA is mixed with 1X

di-deoxyribonucleotides, 100X deoxyribonucleotides and DNA polymerase. Fragments of different sizes

depending on the site of termination are produced and undergo capillary electrophoresis. The relative

positions of these fluorophore labelled fragments in capillary electrophoresis indicate the DNA sequence

when laser detection visualises the fluorophore on the labelled fragments.

Basic Local Alignment Search Tool (BLAST) is used to compare the sequenced DNA and the sequence of

GST in the database. Matching sequence between the two confirms Strain C as the GST gene.

(4)100ml lysis buffer (50mM Tris, 100mM NaCl, & 0.01% (w/v) SDS)TrisMr: 121.14 g/mol.NaClMr: 58.44 g/mol.

Mass of Tris neededTrismole: 50mM x (100/1000) = 0.005mol.Trismass: 0.005mol. x 121.14g/mol.= 0.6057 g

Mass of NaCl neededNaClmole: 100mM x (100/1000) = 0.01mol.NaClmass: 0.01mol. x 58.44 g/mol.= 0.5844 g

Mass of SDS neededSDSvolume: 100ml x 0.01% = 0.01mlSDSmass: 0.0100g

Double distilled wateradded to 0.6057 g Tris, 0.5844 g NaCl & 0.0100g SDS to adjust volume to 100ml.Mixing required

Fig. 1

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(5)Heal afety(a)R agent / Procedures

. IPTG insolution2. Taq Pol erase3. Microwave4. E. coli cultures5. Sybr Safe DNA Dye

(b)Risks. Harmfulifswallowed,in contact wit skin, orinhaled[1]

2. Storedin 5 glycerol[2]. Repeated contact causesdehydration ofskin[3]3. Long term exposure to microwave is carcinogenic. Improperhandling of microwaved item leads to

skin burn

4. E. coilispathogenic. Ingestion causespoisoning,possibly bloody diarrhea[4]5. Exposure can cause mutagenesis ofhuman DNA

(c)Precautions1. Wearglovesto preventskin contact with IPTG2. Studentsarenotallowedto handle Taq Polymerase. Gloves worn when Taqishandled3. Microwavedoorisnot openeduntiltheprocess ofmicrowaving is complete. Heat resistancegloves

areusedto handle microwaveditem

4. E. coil isgenetically modified to benon-pathogenic. Caps of the cultures closed when the culturesareunused. Separatedisposal ofE. coilapparatusto preventspread ofpathogen

5. Safety glassesare worntopreventexposure ofSybr SafeReference:

[1] Safety Data SheetMC4

2 IPTG. Lab M Ltd.

[2]Material Safety Data Sheet(Taq Polymerase). Tepnel LifecodesMolecular Diagnostics.

[3]Material Safety Data Sheet(Glycerol). Tepnel LifecodesMolecular Diagnostics.