Cloning a DNA segment from bacteriophage lambda

1
Cloning a DNA segment from bacteriophage lambda
Recombinant DNA transformed into bacterial
cells Preparation of X-gal plates - by Dr. Soukup
before lab Preparation of competent cells - by
Dr. Soukup before lab DURING LAB Electrophoretic
analysis of restriction digests Transformation
of recombinant plasmid into bacteria Plating of
bacteria onto agar plates ampicillin X-gal
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Cloning a DNA segment from bacteriophage lambda
Electrophoretic analysis of restriction
digests Pour agarose gel with ethidium
bromide Load restriction digests on gel with size
standards Examine results Agarose gel separates
larger DNA molecules by size Ethidium Bromide
fluoresces under UV light EB intercalates into
DNA Put gel on UV light source after
electrophoresis
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Cloning a DNA segment from bacteriophage lambda
Recombinant DNA transformed into bacterial
cells Transformation of recombinant plasmid into
bacteria (cells that take up plasmid are
transformed)
4
Cloning a DNA segment from bacteriophage lambda
Recombinant DNA transformed into bacterial
cells Plasmid characteristics - small circular
double-stranded DNA, usually not necessary for
survival BUT can carry genes that confer
resistance to antibiotics or allow survival in
certain environments
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Cloning a DNA segment from bacteriophage lambda
Recombinant DNA transformed into bacterial
cells Plasmid characteristics Ampicillin
antibiotic used to kill bacteria by interfering
with synthesis of bacterial cell wall and leads
to lysis of bacteria Ampicillin is a
broad-spectrum semi-synthetic penicillin that
will kill gram-negative and gram-positive
bacteria, includes E.coli and Salmonella E.coli
in nature can become resistant to ampicillin by
taking up plasmids that contain Amp-resistant
genes Today we will make one of these plasmids -
ampicillin resistance gene codes for
Beta-lactamase (penicillinase) that inactivates
(degrades) ampicillin
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Cloning a DNA segment from bacteriophage lambda
Recombinant DNA transformed into bacterial
cells Making of competent cells Treat bacterial
cells (E. coli strain DH5?) with CaCl2, which
will make them COMPETENT to take up plasmid DNA
(plasmid DNA will enter the cell) CaCl2 causes
small holes to form in the cell membrane that DNA
can then traverse through We have pre-made
competent cells LABORATORY PROCESS TO MAKE
COMPETENT CELLS Grow small culture of bacterial
from a single colony overnight at 37 C Next day
use small culture to seed large culture and grow
to mid-log phase growth Wash the cells with
CaCl2 Incubate the cells at 4 C for 12 hours
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Cloning a DNA segment from bacteriophage lambda
Recombinant DNA transformed into bacterial
cells Making of competent cells Bacterial growth
in liquid media Exponential growth occurs until
no O2 left Measure cell growth by cell count
(microscope) cell mass (A600)
Doubling time 20 min
lt 90 min
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Cloning a DNA segment from bacteriophage lambda
Recombinant DNA transformed into bacterial
cells Transformation procedure 1. Cells
plasmid DNA - incubate on ice for 20 min (cells
starting to take up plasmid) DO NOT VORTEX OR
ROUGHLY FLICK TUBE WITH CELLS - THEY ARE VERY
FRAGILE 2. Transfer tube to 37 C for 5 min
(heat shock - causes faster uptake of
plasmid) 3. Add nutrient broth (media) without
ampicillin and incubate at 37 C for 45 min USE
STERILE TECHNIQUES!!!! MUST HAVE FLAME
ON!!! During this 45 min the plasmid has time to
start expressing the amp-resistance gene and the
bacteria cell recovers from CaCl2 treatment
(repairs its membrane)
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Cloning a DNA segment from bacteriophage lambda
Recombinant DNA transformed into bacterial
cells Transformation procedure 4. Plate cells
onto agar plates ampicillin X-gal USE STERILE
TECHNIQUES!!!! MUST HAVE FLAME ON!!! You will
be spreading your bacteria onto the agar plate
using a glass rod Pipet culture onto plate and
then spread using STERILE TECHNIQUES!! Dr.
Soukup will demonstrate how to produce single
colonies of control plasmids
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Cloning a DNA segment from bacteriophage lambda
Recombinant DNA transformed into bacterial
cells Transformation procedure 4. Plate cells
onto agar plates ampicillin
X-gal Controls E.coli-pUC18 negative
control Should only get blue colonies E.coli-pUC
18-satellite positive control Should only get
white colonies
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Cloning a DNA segment from bacteriophage lambda
Recombinant DNA transformed into bacterial
cells Transformation procedure 4. Plate cells
onto agar plates ampicillin X-gal Plasmid
also has Lac Z gene which codes for
?-galactosidase (hydrolyzes lactose, other
?-galactosides and X-gal) X-gal is
5-bromo-4-chloro-3-indolyl-?D galactoside -
chromogenic substrate because its product is
colored X-gal converted to blue product by
?-galactosidase pUC18 has a portion of Lac Z
gene, remaining portion is encoded by E.coli
strain SO when cells transformed with pUC18
Complementation occurs and cells make active
?-gal Active ?-gal causes blue colonies to be
produced on agar with X-gal CAN DETERMINE
WHICH PLASMIDS HAVE FOREIGN DNA Polylinker is
inserted in Lac Z gene - if no foreign DNA
inserted then ?-gal made and colonies BLUE If
foreign DNA (bacteriophage DNA) inserted -
complementation is destroyed and no active ?-gal,
so colonies WHITE
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Cloning a DNA segment from bacteriophage lambda
Recombinant DNA transformed into bacterial
cells Transformation procedure 4. Plate cells
onto agar plates ampicillin X-gal Possible
reasons for WHITE COLONIES??? RESTRICTION
DIGESTS RECIRCULARIZATION OF pUC18 during
ligation with no foreign DNA inserted
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Cloning a DNA segment from bacteriophage lambda
Recombinant DNA transformed into bacterial
cells Safety WASH YOUR HANDS WITH
SOAP!!!! DISINFECT LAB BENCH WITH BLEACH OR
ETHANOL SOLUTION IF YOU SPILL BACTERIA TELL DR.
SOUKUP LIMIT EXPOSE OF BACTERIA TO AIR PLACE
ALL BACTERIAL WASTE IN RED BIOHAZARD BAGS WEAR
GLOVES!!