Lambda vectors and their replication
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Enzymes needed:
1.Restriction enzymes: cuts the DNA at specific
sequences to generate a set of fragments
2. DNA ligase: inserts DNA restriction fragments
into replicating DNA molecules to produce
recombinant DNA
Lambda vectors
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Vector types:
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Vector Insert size
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What determines choice of vector?
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-Central 1/3 is the “stuffer” fragment.
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fragment is put in, this keeps the vector at a correct size
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Origin of
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Brock Biology of Microorganisms, 9
th
Edition
(2000)
Prentice Hall, Madigan, Martinko, Parker
steps in cloning with λ :
–
Isolate vector DNA and gene of
interest
–
Cut both with restriction
enzyme(EcoRI)
–
Connect two fragments of
foreign DNA with DNA ligase.
(recombinant DNA)
–
Package DNA by adding cell
extracts containing head and
tail proteins
–
Transfer recombinant
molecules into host cell
(transform)
–
Grow/select transformants:
check recombinant phage for
the presence of desired foreign
DNA sequence by observing
its genetic properties.
Molecular Biology of the Cell, 3
rd
Edition, Garland Publishing, Inc. 1983
http://www-
micro.msb.le.ac.uk/224/Phages.html#Lambda
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Life cycle of lambda
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How do cells leave lysogeny cycle
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Which proteins determine the
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DNA lambda replication
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DNA Replication, W.H. Freeman and Co. (1992)
Kornberg,A.
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