SCIE1106 Lecture Notes - Lecture 16: Multiple Cloning Site, Dna Ligase, Electroporation
L16 Intro to cloning
•
Know properties of r.e
•
Know r.e- digested DNA with complementary
ends can anneal
•
DNA ligase
•
Properties of a plasmid vector + 3 properties
that make them useful in molecular cloning
•
Steps in inserting DNA fragment to plasmid +
amplifying it in bacteria
•
Agarose gel electrophoresis
•
Characteristics of expression vector that make it
useful for large scale proteins production
Recomb DNA molecule – DNA molecule that has
originated from 2/more DNA fragments that are
not found together in nature
Cloning – production of identical copies of a
particular DNA molecule. Isolation of a particular
piece of DNA from the rest of cells DNA
R.E
• Genus species → EcoRI, Escherichia coli,
HindIII, Hemophilus influenza
• Are endonucleases/ endoDNases
o Endo – cuts DNA within/ inside DNA
molecule
• Digest ONLY dsDNA (DNA not RNA) at
internal phosphodiester bonds
• Cuts DNA at specific sites
o Sites that are defined by nt
sequences
o r. sites / r sequences
o cut palindromes (same sequence
from 5 to 3 on BOTH strands )
o cut sites with 4-8 nt
• leave 3OH / 5P on both strands
• leave blunt ends / sticky(cohesive) ends
o 5 overhangs
o 3 overhangs
• complementary base pairs can anneal
together (like for sticky ends/cohesive
ends) but not for blunt ends
R.E s function in nature
1. protect cells from invaders (from viruses)
2. how? - act together with methylase
a. once the DNA is methylated,
enzymes cant get in and cut
DNA ligase
• fills in gaps in sugar phosphate backbones of
annealed DNA strands
o ie, after annealing (pairing), ligase
glues them together
• covalently links 3OH and 5P of fragments
• ATP works with DNA ligase
Plasmids = Vectors
• Are small, ds, circular DNA molecules in
bacteria , is distinct from bacterial
chromosome vectors
o Ie in a bacterial cell you can see
chromosomes and plasmids
distinctly /easily
Plasmid + 3 properties (or 3 regions)
1. ORI – origin of replication , recognized by
bacterial cells replication machinery, allows
many many copies of plasmids in the
bacterial cell
2. Antibiotic resistance marker (Amp) – if a
bacterial cell contains this plasmid, this cell
will be resistance to antibiotic
3. Multiple cloning site / polylinker (SP)– lots
of r.e sites in that region of plasmid (when
exposed to r.e, r.e cuts that site)
Then, DNA fragments and the plasmid that were cut
with the same restriction enzyme anneal together
and DNA ligase glues it together → we have a DNA
recombinant molecule now
Transformation of Bacterial cells
• Introducing the recombinant DNA into the
bacterial cell at ORI
• by heat shock / electroporation
o so then the bacterial cells become
permeable to DNA
o electroporation – weakens the
membrane with short burst of
current
• cells that dont take up plasmid die on
ampicillin plates
• independent plasmid replication occurs
• cell multiplication occurs
• now, there are colonies of cells, where each
cell contains copies of the same
recombinant plasmid
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Recomb dna molecule dna molecule that has. Cloning production of identical copies of a piece of dna from the rest of cell(cid:495)s dna particular dna molecule. R. e: genus species ecori, escherichia coli, are endonucleases/ endodnases. Plasmid + 3 properties (or 3 regions) bacteria , is distinct from bacterial: are small, ds, circular dna molecules in. Then, dna fragments and the plasmid that were cut with the same restriction enzyme anneal together and dna ligase glues it together we have a dna recombinant molecule now. Column chromatography ( to get purified proteins: (host cell extract) with substances inside a test tube. Overexpressed proteins have his-tags: bind to metal ions (ni2+, zn2+, eluted from column, by reducing ph of solvent. Requirements: agarose red algal carb, heated in buffer to, molecular size standards that is separated on the gel, used to determine size of dna fragments dissolve ( needs to be in aq phase, dna restriction fragments.
