Download Human Mitochondrial DNA

DNA Ligation & Colony Transformation
Carolina Kit
Isabelle Muschamp
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The plasmid vector must be cut with a
restriction endonuclease (aka: restriction
enzyme)
DNA ligase joins the DNA fragment & vector
DNA
Host cell is made competent so can plasmid can
enter
Transformed cells are grown on selection media
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Extrachromosomal
DNA molecules
Small; varies from 1 to
over 1,000 kilobase pairs
Circular
Usually carry one or a few
genes
Replicate separately from the
chromosome
A type of cloning vector used to
carry a gene not found in the
bacterial host’s chromosome
Treatment of plasmids pAMP and pKAN with a mixture of BamHI and
HindIII are given for the Easy Gene Splicer lab
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4539 base pairs
a single replication origin
a gene (ampr)conferring resistance to the antibiotic ampicillin (a relative of
penicillin)
a single occurrence of the sequence
5' GGATCC 3'
3' CCTAGG 5‘
that, as we saw above, is cut by the restriction enzyme BamHI
a single occurrence of the sequence
5' AAGCTT 3'
3' TTCGAA 5‘
that is cut by the restriction enzyme HindIII
Treatment of pAMP with a mixture of BamHI and HindIII produces:
-a fragment of 3755 base pairs carrying both the ampr gene and the
replication origin
-a fragment of 784 base pairs
-both fragments have sticky ends
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4207 base pairs
a single replication origin
a gene (kanr) conferring resistance to the antibiotic
kanamycin
a single site cut by BamHI
a single site cut by HindIII
Treatment of pKAN with a mixture of BamHI and
HindIII produces:
-a fragment of 2332 base pairs
-a fragment of 1875 base pairs with the kanr gene
(but no origin of replication)
-both fragments have sticky ends
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Both the DNA that will be inserted and the
plasmid DNA are ligated together
Sticky ends help this process by stabilizing
the pieces together with base-pairing, but the
enzyme DNA ligase is added to the reaction
to cause the DNAs to become covalently
linked together
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After ligation of the insert DNA and your
plasmid, the new chimeric (an organism that
is composed of genetically different tissues,
either naturally or as a result of a laboratory
procedure) of DNA into bacteria
Transformation of the bacteria with
recombinant DNA is easily performed by
shocking the cells
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The process of transferring foreign DNA fragments into a
recipient (host) cell for growth and replication
Scientists begin this process by fusing two different sets of
DNA together creating a molecule of recombinant DNA or
rDNA. This particular molecule is the product of the gene
of interest (the desired gene) and another source of
plasmid DNA coming together through the use of a
restriction enzyme, which cuts the DNA at a specific
nucleotide sequence, and a ligase which binds the
separate strands of DNA into one form.
After the rDNA is made, the cells which are going to be
used as a vehicle for transformation are made
“competent” or prepared to receive foreign DNA.
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The cells are concentrated into a pellet through the use of
a centrifuge, and their membranes are made porous so
that the rDNA has a route to enter the cell.
The rDNA is added to the cell culture and some of the
rDNA plasmids are absorbed, but to increase their
absorption numbers the culture undergoes a heat/cold
shock. The hot water bath enlarges the cell’s pores and
more plasmids are “sucked” in. The culture is then quickly
transferred to the ice which traps the plasmids within the
cell’s membrane.
Cells containing the foreign DNA grow and multiply within
the tube, but to ensure that transformation was successful
and purification of the gene of interest to proceed, the
culture is grown on mediums so visual confirmation can be
made.
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Endonucleases:
◦ in nature, they protect
bacteria from intruding DNA
◦ cut up (restrict) the viral DNA
◦ cut only at very specific
nucleotide sequences
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Restriction site:
recognition sequence for
a particular restriction enzyme
Restriction fragments:
segments of DNA cut by
restriction enzymes in a
reproducible way
DNA ligase:
joins the sticky ends of DNA
fragments
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rDNA (recombinant DNA)—the produced piece of DNA from inserted
another piece of DNA
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Recognize specific sites to cut the DNA
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Blunt ends—straight across
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Sticky ends—one side of DNA is longer than the other, these
overhangs allow for complementary matches between two DNA
pieces cut by the same enzyme, the sticky ends match and pasting
may occur to produce an rDNA molecule
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More than 1200 restriction enzymes discovered & isolated from
bacteria
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Read 5 3
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Palindromic (example radar or
GAATTC
CTTAAG
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Transformation – the uptake and expression of foreign DNA by a cell
Transduction – the use of viruses to transform or genetically engineer
cells
Competent/competency – the ability of cells to take up DNA
Selection – the process of screening potential clones for the expression of
a particular gene, for example, the expression of a resistance gene (such as
resistance to ampicillin) in transformed cells
Transformation efficiency – a measure of how well cells are transformed
to a new phenotype
Recovery period – the period following transformation where cells are
given nutrients and allowed to repair their membranes and express the
“selection gene(s)”
Beta-galactosidase gene – a gene that produces beta-galactosidase, an
enzyme that converts the carbohydrate X-gal into a blue product
Green fluorescent protein – a protein found in certain species of jellyfish
that glows green when excited by certain wavelengths of light
(fluorescence)
Scale-up – the process of increasing the size or volume of the production
of a particular product
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Week Before: read lab & Flowchart, only 6
groups
Thursday—Lecture, pre-quiz
Monday—part A
Tuesday—part B
Thursday--part C
Friday FIRE—come view results
Prepare
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LB agar plates (<1 month before)
2 LB agar plates for streaking to use in part B
16 (gives 4 extra) LB agar plates for part C
12 (gives 4 extra) LB agar plates with ampicillin and kanamycin
coming with kit—check for 2 LB/amp and 2 LB/kan for control group
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Aliquot for 6 groups
Part A
--20uL Ligation Buffer/ATP/ligase (6)
--10uL pAMP (5 tubes—pKAN control does not get one)
--10uL pKAN (5 tubes—pAMP control does not get one)
Part B
--500uL Calcium Chloride (6)
--500uL LB (6)
Locate Supplies:
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Spreaders
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loops
set up for part A
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ice
set up for part B
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Streak starter plates (12-20 hours before part B) (start after school)
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42C water bath
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37C incubator
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put calcium chloride on ice
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When finish store at 0C (beaker of ice in fridge)
set up for part C
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37C incubator
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2 lab station groups will be control groups:
1. -–pAMP + ligase (use 10uL water)
2. --pKAN + ligase (use 10uL water)
Follow same directions, but run these instead
of pAMP/KAN
Only plate on LB/amp and LB/kan plate (do
not spread on LB plates)
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Group 1—pAMP control
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Group 2—pKAN control
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Group 3
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Group 4
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Group 5
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Group 6
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http://bioinformatics.dnalc.org/gmo/animation/g
mo.html
http://www.carolina.com/text/teacherresourc
es/instructions/biotech/ez_gene_splicer_kit.p
df
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Endonucleases – enzymes that cut RNA or
DNA at specific sites; restriction enzymes are
endonucleases that cut DNA
Sticky cells – restriction fragments in which
one end of the double stranded DNA is longer
than the other; necessary for the formation of
recombinant DNA
Restriction enzyme mapping – determining
the order of restriction sites of enzymes in
relation to each other
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Samples of the plasmid fragments are
mixed with DNA ligase and incubated at
room temperature for 2-24 hours.
Complementary BamHI and HindIII “sticky
ends” hydrogen-bond to align restriction
fragments. Ligase catalyzes the formation
of phosphodiester bonds that covalently
link the DNA fragments to form stable
recombinant DNA molecules.
Use micropipets (instead of transfer)
After 2-24hours, will store in fridge at 4C
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Transform E.coli with the ligated plasmid
DNA. E. coli cells are scraped off an LB agar
plate and suspended in two tubes
containing solution of calcium chloride.
The ligated pAMP/KAN plasmid is added to
one cell suspension, and both tubes are
incubated at 0C, then heat shocked at 42C,
cooling and addition of LB broth, cells then
recover.
I will store cells at 0C (beaker of ice in
fridge) after 5-6 hours of incubation
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Samples of the cells are plated on two types
of growth media: plain LB agar and LB agar
with ampicillin and kanamycin. Incubate to
grow cells. Only cells with both ampicillin
and kanaycin will be expressed on the 2nd
plate
Come in at FIRE to see
You are receiving 2 LB plates—so you can do
both plates in PART C step 1.C and 2
Use spreaders at Part C 3