(1) PCR experiment

Key
M
100bp Molecular marker
Ax
Strain A with Primer X PCR
By
Strain 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 C x & Dy lanes due to
mispriming of X&Y to C&D in PCR

(2) Summary Table

E. coli strain tested

Test performed A B C D E F
(3)
Ampicillin R R R S R S
Resistant (R) or
Sensitive (S)?
Kanamycin S S S R R S
Resistant (R) or
Sensitive (S)?
Immunodetection Negative Negative Strong Negative Negative Negative
with anti-GST
Negative, weak
or strong
Size of PCR 900bp N/D N/D N/D N/D N/D
product with
primer pair X
Size of PCR N/D 500bp N/D N/D N/D N/D
product with
primer pair Y
Fluorescence Not Not green Not Green N/D N/D
Microscopy Green green
Green
fluorescence or
not
Gene identity Small GTP An N/D N/D N/D N/D
from Binding Protein antibody
bioinformatics ρA
analysis
Identity of Small GTP An GST GFP Kanamycin None
cloned gene Binding Protein antibody Resistance
product ρA
Further experiment
Strain 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.
Fig. 1

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. 95°C 5mins (dsDNA to ssDNA denaturation)
2. 95°C 30sec (dsDNA to ssDNA denaturation)
3. 55°C 30sec (Primer annealing)
4. 75°C 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 3’OH 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 needed

Trismole: 50mM x (100/1000) = 0.005mol.
Trismass: 0.005mol. x 121.14 g/mol.= 0.6057 g

Mass of NaCl needed

NaClmole: 100mM x (100/1000) = 0.01mol.
NaClmass: 0.01mol. x 58.44 g/mol.= 0.5844 g

Mass of SDS needed

SDSvolume: 100ml x 0.01% = 0.01ml
 SDSmass: 0.0100g

Double distilled water added to 0.6057 g Tris, 0.5844 g NaCl & 0.0100g SDS to adjust volume to 100ml.
Mixing required
(5) Health & safety
(a) Reagents/ Procedures
1. IPTG in solution
2. Taq Polymerase
3. Microwave
4. E. coli cultures
5. Sybr Safe DNA Dye

(b) Risks
1. Harmful if swallowed, in contact with skin, or inhaled[1]
2. Stored in 50% glycerol[2]. Repeated contact causes dehydration of skin[3]
3. Long term exposure to microwave is carcinogenic. Improper handling of microwaved item leads to
skin burn
4. E. coil is pathogenic. Ingestion causes poisoning, possibly bloody diarrhea[4]
5. Exposure can cause mutagenesis of human DNA

(c) Precautions
1. Wear gloves to prevent skin contact with IPTG
2. Students are not allowed to handle Taq Polymerase. Gloves worn when Taq is handled
3. Microwave door is not opened until the process of microwaving is complete. Heat resistance gloves
are used to handle microwaved item
4. E. coil is genetically modified to be non-pathogenic. Caps of the cultures closed when the cultures
are unused. Separate disposal of E. coil apparatus to prevent spread of pathogen
5. Safety glasses are worn to prevent exposure of Sybr Safe

Reference:
[1] Safety Data Sheet MC402 IPTG. Lab M Ltd.
[2] Material Safety Data Sheet (Taq Polymerase). Tepnel Lifecodes Molecular Diagnostics.
[3] Material Safety Data Sheet (Glycerol). Tepnel Lifecodes Molecular Diagnostics.