what are restriction and modification emzymes?

1) single-stranded "sticky" ends
2) methylated DNA not cut

Agarose gel electrophoresis
tell me about everything

DNA migrates toward positive electrode

what does restriction endonucleases do?

selectively cleave sites of DNA

Steps of gene cloning

1) isolate DNA to be cloned
2) use a restriction enzyme or PCR to generate fragments of DNA
3) generate a recombinant molecule by inserting DNA fragments into a
cloning vector (DNA ligase)
4) introduce recombinant molecule into new host which will express
the gene

what kind of cut would restriction endonuclease make

staggered cut

where does restriction endonuclease cut

at recognition site

what's PCR stands for

Polymerase chain reaction

Use of PCR in gene cloning

1) isolate cellular genome
2) generate mixture of DNA fragments
3) obtain band containing desired fragment
4) isolate DNA fragment
5) construct recombinant vector
6) isolate recombinant clone

what do you need in order to move to step 2 ' generate mixture of DNA
fragment' from isolate cellular genome (step 1)?

treatment with restriction enzyme

what do you need from step 2 to step 3?

agarose gel electrphoresis
southern blotting

what d you need from step 3 (obtain band containing desired fragment)
to step 4 (isolate DNA fragment)?

extraction of DNA
electrophoresis on a different gel

what's needed in the process going to step 5 (construct recombinant
vector) from step4 (isolate DNA fragment)?

1) anneal with plasmid or phage vector
2) DNA ligase treatment

what enzymes terminates DNA synthesis

ddNTP

what is the last thing that we need to isolate recombinant clone?

transform bacterial host and culture bacteria

What's significant about ddNTP?

there are hydrogens on 2' and 3' of pentagen

what's needed in order to make DNA + niPP?

DNA polymerase and DNA template

what's the original sanger sequencing method?

DNA sequencing

steps of DNA sequencing

1) isolated unknown DNA fragment
2) DNA is denatured to produce single template strand
3) Labeled specific primer molecule hybridizes to the DNA strand
4) DNA polymerase and regular nucleotide mixture are added
5) newly replicated strands are terminate at the point of addition
of a dd nucleotide
6) schematic view of how all possible positions on the fragment are
occupied by a labeled nucleotide

what is the more common method that people use

4 ddNTPs labeled with different colored fluors to terminate reactions.
It can be run in a single lane

what is the example of genetic enginnering of plants

agrobacterium tumefaciens and crown gall disease