1. a. Gateway cloning allows for directional cloning of pieces of
DNA. How should one create the sequence fragment so that
it can be used in the Gateway system?
Het heft een recognition site nodig die herkend kan worden door
recombinase waardoor de sequentie van interesse ‘’geflipt’’ kan
worden naar de entry clone.
The BP reaction, PCR with a primer that is specific for your target of
interest with a tail. With the PCR you will amplify the region of
interest and you will add the attp sites. Using the donor vector you
can flip them to the entry clone
So PCR based.
b. Make an overview of the enzymes needed for classical
molecular cloning,
Gibson assembly, and Gateway cloning. How do they
compare?
Classical molecular cloning: type II restriction enzymes (which
recognize and cut directly within the recognition site)
Gibson cloning: DNA polymerase, ligase, nuclease
Gateway cloning: recombinase (restriction enzyme + ligase)
Classical: restriction enzymes, DNA ligases (+Pol or Exo, to blunt)
Gibson: cocktail of Pol, 5’exo and DNA ligase
Gateway cloning: recombinase (restriction enzyme + ligase)
Gibson cloning is without restriction enzymes and classical molecular
cloning + gateway cloning is with the use of restriction enzymes. At
Gibson you have overhanging ends and with the use of exonuclease
can be turned into sticky ends.
Practical points: difference in buffers needed, incubation
temperatures, phosphorylation status, methylation status, etc.
c. Is it possible to use the Gateway system for the
generation of fusion protein constructs?
Yes but you have to be aware that there will still be att sites present,
meaning that infront of after your gene, the recognition sites will still
be there. You have to consider reading frames that ‘run’ over the
attp sites.
2. For each type of transfection method (chemical, physical,
biological)
a. list two common examples
b. indicate their main application
c. decide where they reside on the ‘transient versus stable’
axis and whether they may damage the host genome. It
depends on what happens in the nucleus with those DNA fragments.
If the cell is having homology arms and it is driving homology
directed repair then it is stable. If it is linear it is more prone to
become stable. If it is a plasmid it needs to be opened up before you
DNA. How should one create the sequence fragment so that
it can be used in the Gateway system?
Het heft een recognition site nodig die herkend kan worden door
recombinase waardoor de sequentie van interesse ‘’geflipt’’ kan
worden naar de entry clone.
The BP reaction, PCR with a primer that is specific for your target of
interest with a tail. With the PCR you will amplify the region of
interest and you will add the attp sites. Using the donor vector you
can flip them to the entry clone
So PCR based.
b. Make an overview of the enzymes needed for classical
molecular cloning,
Gibson assembly, and Gateway cloning. How do they
compare?
Classical molecular cloning: type II restriction enzymes (which
recognize and cut directly within the recognition site)
Gibson cloning: DNA polymerase, ligase, nuclease
Gateway cloning: recombinase (restriction enzyme + ligase)
Classical: restriction enzymes, DNA ligases (+Pol or Exo, to blunt)
Gibson: cocktail of Pol, 5’exo and DNA ligase
Gateway cloning: recombinase (restriction enzyme + ligase)
Gibson cloning is without restriction enzymes and classical molecular
cloning + gateway cloning is with the use of restriction enzymes. At
Gibson you have overhanging ends and with the use of exonuclease
can be turned into sticky ends.
Practical points: difference in buffers needed, incubation
temperatures, phosphorylation status, methylation status, etc.
c. Is it possible to use the Gateway system for the
generation of fusion protein constructs?
Yes but you have to be aware that there will still be att sites present,
meaning that infront of after your gene, the recognition sites will still
be there. You have to consider reading frames that ‘run’ over the
attp sites.
2. For each type of transfection method (chemical, physical,
biological)
a. list two common examples
b. indicate their main application
c. decide where they reside on the ‘transient versus stable’
axis and whether they may damage the host genome. It
depends on what happens in the nucleus with those DNA fragments.
If the cell is having homology arms and it is driving homology
directed repair then it is stable. If it is linear it is more prone to
become stable. If it is a plasmid it needs to be opened up before you
