SSA 1-2 – Molecular Cloning
Questions
1. a. What is a plasmid?
A plasmid is a small circular DNA molecule that are used as carriers of DNA, and they can
replicate independently.
A circular piece of DNA which has an origin of replication and they will be replicated by the
bacteria. The promotor is needed if you want to express a gene and initiate transcription
b. Which modifications were introduced in natural plasmids to make them useful lab tools?
They were made non-conjugating to prevent horizontal transfer. Natural plasmids in bacteria
can make a bridge and then copy and inject the plasmid in the other bacterium and then it
has obtained a copy. You don’t want this in the lab because then you cant control which
bacteria has the plasmid or not. We want to be able to decide if the plasmid is able to enter
the bacteria. And you cant control the clonality and that’s why they made them non-
conjugating
2. a. Is it possible to ligate a DNA fragment that was generated by BamH-I into a plasmid
vector that was linearized using Bgl-II ?
Omdat allebei de sticky ends dus de overhangs de sequentie hebben van GATC is het wel
mogelijk, want die kunnen ligeren aan elkaar.
Yes because the exact same overhang will fit the overhang of the other one
b. If yes, would it be possible to digest the resulting sequence with any of these two
enzymes?
Nee het is niet meer mogelijk om een digestie uit te voeren omdat BamHI de sequentie 5’-
GGATCC-3’ herkend en niet de sequentie 5’-AGATCT-3’ dus ook al waren de ends hetzelfde
de rest van de sequentie is niet hetzelfde waardoor het fragment er niet meer uit gehaald
kan worden door de andere restrictie enzym, want deze wordt niet herkend.
The recognition site will be different so you cant redigest them because the recognition site
will be 5’-GGATCT-3’ and this wont be recognized
3. The figure below depicts a schematic ‘map’ of the gene that encodes the protein MIN13.
The approximate binding spots for three oligonucleotide primers are included. Based on
this, predict the length of the amplicon(s) that will be generated
It is important to see if the template is genomic DNA or cDNA!!!! Genomic DNA contains
introns and cDNA doesn’t so only the exons are present and this will influence the length of
the amplicons that are generated !!!!!!!!!!
a. using primers 1 and 2 on this genomic DNA segment
Questions
1. a. What is a plasmid?
A plasmid is a small circular DNA molecule that are used as carriers of DNA, and they can
replicate independently.
A circular piece of DNA which has an origin of replication and they will be replicated by the
bacteria. The promotor is needed if you want to express a gene and initiate transcription
b. Which modifications were introduced in natural plasmids to make them useful lab tools?
They were made non-conjugating to prevent horizontal transfer. Natural plasmids in bacteria
can make a bridge and then copy and inject the plasmid in the other bacterium and then it
has obtained a copy. You don’t want this in the lab because then you cant control which
bacteria has the plasmid or not. We want to be able to decide if the plasmid is able to enter
the bacteria. And you cant control the clonality and that’s why they made them non-
conjugating
2. a. Is it possible to ligate a DNA fragment that was generated by BamH-I into a plasmid
vector that was linearized using Bgl-II ?
Omdat allebei de sticky ends dus de overhangs de sequentie hebben van GATC is het wel
mogelijk, want die kunnen ligeren aan elkaar.
Yes because the exact same overhang will fit the overhang of the other one
b. If yes, would it be possible to digest the resulting sequence with any of these two
enzymes?
Nee het is niet meer mogelijk om een digestie uit te voeren omdat BamHI de sequentie 5’-
GGATCC-3’ herkend en niet de sequentie 5’-AGATCT-3’ dus ook al waren de ends hetzelfde
de rest van de sequentie is niet hetzelfde waardoor het fragment er niet meer uit gehaald
kan worden door de andere restrictie enzym, want deze wordt niet herkend.
The recognition site will be different so you cant redigest them because the recognition site
will be 5’-GGATCT-3’ and this wont be recognized
3. The figure below depicts a schematic ‘map’ of the gene that encodes the protein MIN13.
The approximate binding spots for three oligonucleotide primers are included. Based on
this, predict the length of the amplicon(s) that will be generated
It is important to see if the template is genomic DNA or cDNA!!!! Genomic DNA contains
introns and cDNA doesn’t so only the exons are present and this will influence the length of
the amplicons that are generated !!!!!!!!!!
a. using primers 1 and 2 on this genomic DNA segment
