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
what kind of cut would restriction endonuclease make
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
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
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?
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