BIO1022 Lecture Notes - Lecture 4: Intron, Dna Ligase, Restriction Enzyme
BIO1022 – Lecture 4 – Week 2
- technique 1 - restriction enzymes - to digest (cut) DNA
- separating DNA restriction fragments by gel electrophoresis
- technique 2 - plasmids - cloning vectors
- molecular cloning
• using the cell’s own tools to answer experimental questions
• extract DNA - genetic DNA
• cut the DNA with a restriction enzyme - recognises specific
sequences and cuts
• mix the digested DNA with a cloning vector - cut with the same
restriction enzyme
• add DNA ligase
• transform these recombinant plasmids into bacteria
•
- separating DNA restriction fragments by gel electrophoresis
• DNA is negatively charged
- plasmids - cloning vectors
• small circular DNA molecules
• provide a way for bacteria to rapidly acquire or lose genes
• can use an enzyme to cut open an enzyme
• insert gene fragments into these cloning vectors
- transformation of bacteria - storing and growing the plasmid
• bacteria take up DNA from the environment if they are competent
• grow bacteria
• introduce DNA
• put holes in bacteria
- making a genomic library
• what can you do?
•
o tehcni
o
▪ DNA sequencing
▪
▪ dideoxy chain termination
▪ template strand of DNA
▪ warming up so two strand separate
▪ primer sequence complementary to sequence
- hybridisation with a nucleic acid probe
• label probe with radiation
- cloning a cDNA
• a synthetic gene copy without the introns
• extract RNA
• use never transcriptase to turn into synthesis cDNA - complementary
DNA
• mix the cDAN with a cloning vector to create recombinant plasmids
• transform these recombinant plasmids into bacteria
• cDNA library
• contains the coding regions of all expressed genes from that tissue
- retro viruses - infect cells - use those cells to spread virus
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Document Summary
Technique 1 - restriction enzymes - to digest (cut) dna. Separating dna restriction fragments by gel electrophoresis. Technique 2 - plasmids - cloning vectors. Separating dna restriction fragments by gel electrophoresis: dna is negatively charged. Plasmids - cloning vectors small circular dna molecules: provide a way for bacteria to rapidly acquire or lose genes can use an enzyme to cut open an enzyme insert gene fragments into these cloning vectors. Transformation of bacteria - storing and growing the plasmid: bacteria take up dna from the environment if they are competent, grow bacteria introduce dna, put holes in bacteria. Making a genomic library: what can you do? tehcni, dna sequencing, dideoxy chain termination, warming up so two strand separate, primer sequence complementary to sequence template strand of dna. Hybridisation with a nucleic acid probe label probe with radiation. Cloning a cdna: a synthetic gene copy without the introns, extract rna, use never transcriptase to turn into synthesis cdna - complementary.