The
Lambda protocol
National Centre for Biotechnology Education
Copyright © Dean Madden, 2012
www.ncbe.reading.ac.uk
Lambda DNA
Lambda phage
E. coli DNA
E. coli
Lambda DNA enters
cell and forms a ring
Lysogenic cycle
Lytic cycle
Activation
Lambda DNA
integrates within the
host E. coli
chromosome
UV light
Lambda DNA
replicated
Lysis
Copyright © Dean Madden, 2012
E. coli bursts open, releasing
new lambda phages
Head
with DNA
Tail
Non-essential region
Copyright © Dean Madden, 2012
Lambda DNA
48 502 base pairs
BamHI
Copyright © Dean Madden, 2012
BamHI
Copyright © Dean Madden, 2012
EcoRI
Copyright © Dean Madden, 2012
HindIII
Copyright © Dean Madden, 2012
HindIII
2027
23103
564
2322
9416
6557
125
4361
BamHI
5505
16841
5626
6527
7233
6770
EcoRI
21226
4878
5643
7421
5804
3530
Copyright © Dean Madden, 2012
Copyright © Dean Madden, 2012
Summary of procedure
Copyright © Dean Madden, 2012
Microsyringe
HOLD HERE
Do not touch the point!
Graduated tip
10 µL
Copyright © Dean Madden, 2012
2 µL
Mass
A microgram is one millionth of a gram
1 000 micrograms (µg) = 1 milligram (mg)
1 000 milligrams = 1 gram (g)
Volume
A microlitre is one millionth of a litre
1 000 microlitres (µL) = 1 millilitre (mL)
1 000 millilitres = 1 litre (L)
Copyright © Dean Madden, 2012
Cap the
tube firmly
Add 100 µL
of distilled water
Copyright © Dean Madden, 2012
30 µg of dried
lambda DNA
Flick the tube
repeatedly to
dissolve the
DNA
Mix the DNA
solution
thoroughly
before
dispensing it
Lambda
DNA
solution
20 µL
20 µL
20 µL
20 µL
Use a new tip to add the DNA solution
to each tube, to prevent contamination
BamHI
HindIII
Empty
Copyright © Dean Madden, 2012
EcoRI
Close the tubes and push them
firmly into the foam floater
Copyright © Dean Madden, 2012
Incubate for 30–45 minutes at 37°C
Carbon fibre
tissue
42 mm
Cut two
electrodes
Copyright © Dean Madden, 2012
22 mm
Frosted panel
on this side
Molten agarose
55–60 °C
Copyright © Dean Madden, 2012
Copyright © Dean Madden, 2012
Pour buffer over the
gel before removing
the comb
Mix loading dye into
each
DNA sample
2 µL
2 µL
2 µL
2 µL
Copyright © Dean Madden, 2012
TBE buffer solution
2–3 mm depth of
buffer over the gel
Label the end of
the tank to show
the contents of
each well
Black card under
the tank reveals
the wells for
loading
Copyright © Dean Madden, 2012
Electrodes
Copyright © Dean Madden, 2012
Place a comb over the
tank to reduce
evaporation
Direction of DNA
movement
Copyright © Dean Madden, 2012
Azure A stain
Leave the stain on
for 4 minutes only
Copyright © Dean Madden, 2012
Positively-charged Azure A binds
to the negatively-charged
phosphate groups of the DNA
DNA
Copyright © Dean Madden, 2012
Wells
Millimetre graph paper
beneath the gel is a
convenient way to
measure distances
Loading dye
Area with DNA
bands
Copyright © Dean Madden, 2012
Specimen
results
EcoRI HindIII BamHI
Copyright © Dean Madden, 2012
Uncut