national centre for biotechnology education the lambda protocol copyright © dean madden, 2012
TRANSCRIPT
National Centre for Biotechnology Education
www.ncbe.reading.ac.uk
The
Lambda protocol
Copyright © Dean Madden, 2012
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E. coli
Lambda phageLambda DNA
E. coli DNA
Lambda DNA enters cell and forms a ring
Lysogenic cycle Lytic cycle
Activation
UV lightLambda DNA
integrates within the host E. coli
chromosome
Lambda DNA replicated
Lysis
E. coli bursts open, releasing new lambda phages
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Headwith DNA
Tail
Lambda DNA48 502 base pairs
Non-essential region
Head
Tail
Integration of DNA into
bacterial chromosom
eEarly control
DNA synthesisLate control
Lysis of bacterium
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BamHI
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BamHI
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EcoRI
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HindIII
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BamHI
HindIII
EcoRI
23103
2027
2322 9416
564
125
6557
4361
5505 16841 5626 6527 7233 6770
21226 4878 5643 7421 5804 3530
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Summary of procedure
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Microsyringe
Graduated tip
HOLD HEREDo not touch the point!
10 µL
2 µL
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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)
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Add 100 µLof distilled water
30 µg of driedlambda DNA
Cap the tube firmly
Flick the tube repeatedly to dissolve the
DNA
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Mix the DNAsolution
thoroughlybefore
dispensing it
LambdaDNA
solution
20 µL 20 µL 20 µL 20 µL
Use a new tip to add the DNA solutionto each tube, to prevent contamination
EcoRI BamHI HindIII Empty
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Close the tubes and push them firmly into the foam floater
Incubate for 30–45 minutes at 37°C
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Cut two electrodes
Carbon fibretissue
42 mm
22 mm
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Frosted panel on this side
Molten agarose55–60 °C
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Pour buffer over the gel before removing
the comb
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Mix loading dye into each
DNA sample
2 µL2 µL
2 µL 2 µL
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TBE buffer solution
Label the end of the tank to show the contents of
each well
Black card under the tank reveals
the wells for loading
2–3 mm depth of buffer over the gel
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Electrodes
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Direction of DNA movement
Place a comb over the tank to reduce
evaporation
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Leave the stain on for 4 minutes only
Azure A stain
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DNA
Positively-charged Azure A binds to the negatively-charged
phosphate groups of the DNA
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Area with DNA bands
Wells Loading dye
Millimetre graph paper beneath the gel is a convenient way to measure distances
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Specimenresults
EcoRI BamHIHindIIIUncut