Lecture 2 Flashcards
How do you clone DNA
1) Cloning vactor is cleaved with restriction endonuclease (Isolating DNA)
2) DNa fragment of interest is obtained by cleaving chromosome with a restriction endonuclease.
3) Fragments are ligated to the prepared cloning vector. (Recombinant vector)
4) DNA is introduced into the host cell.
5) Propagation (cloning) produces many copies of recombinant DNA.
How do you isolate DNA
A. Source DNA (e.g. E. coli bacteria)
i. Centrifuge: 8,000 rpm to pellet cells
ii. Discard supernatant (media/serum)
iii. Resuspend cells in Tris/EDTA solution
B. Lyse cells to release contents (DNA, RNA & proteins)
Chemical lysis [e.g. add alkaline + detergent (Experiment 2)]
C. Separate DNA
i. Potassium acetate (neutralise solution),
centrifuge (spin)
ii. Transfer supernatant containing plasmid DNA,
add isopropanol to precipitate DNA, spin
iii. Pour off supernatant, add ethanol, spin
D. Analyse isolated DNA for purity
Agarose gel electrophoresis
UV Spectrophotometry
How do you obtain DNA fragment of interest
(i) Use restriction enzymes to ‘cut’ DNA at specific sites
screen for fragment of interest
(ii) Use PCR to amplify gene of interest
b. Choose vector:
Usually a plasmid (small circular DNA molecule,
independently replicating):
‐ Cloning plasmid or Expression plasmid
What are the recognition site
often
palindromic
TaqI: 5’ TCGA 3’
3’ AGCT 5’
How does the sticky end of complementary sequence re-anneal
h-bonding
Phosphodiester bonds can then be formed between broken bonds of adjacent bases by DNA ligase
How does no overhang DNA reanneal
Using adjacent bases – DNA ligase is required for this covalent linkage
What is the use of Mg2+
• Typically require Mg2+ for catalysis e.g. Mg2+ binding site in EcoRV – Mg2+ ion activates/positions water molecule so it can attack phosphate
How toHydrolyze bond between 3’ oxygen and phosphorus
DNA strand with a free 3’ hydroxyl group and another strand with a 5’ phosphoryl group
How do we identify that the DNA has been ‘cut’ with RE?
Use AGAROSE GEL ELECTROPHORESIS: separate restriction
fragments
SOUTHERN BLOT ANALYSIS
identify gene of interest via hybridization
Detect a DNA fragment carrying gene of interest by
- Use sequence‐specific probe complementary to gene of interest
(Synthetic “probe” = oligonucleotide: short sequence of single
stranded DNA) - Hybridize radiolabelled/fluorescent probes to DNA fragment
A critical parameter in hybridization is the
melting point(Tm)
What is Tm
the temperature at which 50% of the double‐
stranded DNA duplex will dissociate to become single stranded
What is southern blotting
Identify gene of interest via specific probe complementary to
gene of interest
What are the steps for SOUTHERN BLOT ANALYSIS
1) Isolated DNA, cut by REs
2)DNA fragments separated by
agarose gel electrophoresis
3) Blotting:
Separated DNA fragments
transferred to nitrocellulose paper
4)Remove nitrocellulose paper
filter from agarose gel, will
contain tightly bound DNA
fragments
5) Hybridisation step: Radioactively‐labelled DNA probe incubated with nitrocellulose filter to hybridised to complementary DNA bands
6)Detection: Nitrocellulose filter exposed to X‐ray film (autoradiography) to detect probe bound to target sequence
