March 6 Flashcards
(9 cards)
What is subcloning?
It is when use restriction enzymes to cut insert out of plasmid, put the insert in a new vector
For the following SUBCLONING methods, what is it, are ends compatible or not, what must be done to them if not. And is the cloning directional? Do need to add phosphatase to prevent vector self-ligation?
Single digest, 1 enzyme
Single digest, 2 compatible enzymes
Single digest, 2 incompatible enzymes
Double digest
Single digest, 1 enzyme: Both insert from plasmid and new vector cut with same single enzyme. Non directional, self ligation possible
Single digest, 2 compatible enzymes: Plasmid insert and vector each digested with diff single enzymes. Ends still compatible as enzymes are compatible. Still non directional, ligation still possible.
Single digest, 2 incompatible enzymes: Like above but ends are incompatible, so insert can’t insert no matter what orientation. Need to blunt the ends to get ligation, so it will still be non-directional, and vector can still self-ligate.
Double digest: Insert from plasmid and new vector both digested with same enzymes or combination. So have 1 end of vector cut with same as one for insert, and another will cut other end of insert and other end of vector. If these cut sites are diff between the enzymes, then cloning is directional and vector self-ligation is not possible
What is PCR cloning?
It is when do PCR on the insert, then ligating it into the vector
There are 3 types of PCR cloning : Blunt, TA,, and RE. What are they, and what is the end result (blunt ends or not, and directional or not)?
Blunt is if proofreading version of Taq Pol is used. So vector must be blunt as well for blunt ended insert. This is non-directional
TA is when the insert after PCR will have 3’ A overhands if Taq is used. Therefore to compensate, vector must have 3’ T overhang. So can have 3’ T added to blunt vector. Also non directional
RE is when use restriction enzyme so insert has sticky ends. Vector will as well, and can be directional or non directional
What is ligase-free cloning? Is it directional?
Don’t use T4 ligase. Needs longer overhangs (15 bp) than used by restriction enzymes. So don’t use MCS, RE cuts somewhere else. Then treat with T4 DNA pol and dNTP for the chosen nucleotide. The T4 DNA pol has 3-5’ exonuclease activity, and will remove nucleotides until it reaches the nucleotide that you added. As 3’ to 5’ exo, it will only remove nucleotides on top strand until reaching the nucleotide. This will give you bigger overhang.
Once all done, anneal insert and vector, then insert into bacteria. They will repair the 4 nicks in the plasmid with their enzymes. This method is directional
What is TOPO cloning? How does it work?
It is when you use topoisomerase that will incorporate the insert into the vector. All need PCR product of insert. This takes about 5 minutes to do.
Have a special vector that is linear and linked to topopisomerase on the 3’ PO4. So need insert with a 5’ OH for this to work so it will bind to 3’ PO4 via topoisomerase.
What is gateway cloning? Is it directional or not, how long does it take?
It is when combine the attachemnt sequences of phage DNA (attP) and bacterial chromosome (attB) to make 2 new sites, left (attL) and right (attR). This works so have one in vector, other in insert, then they combine to make L and R in the final plasmid. This is directional, it takes and hour
What is a Gibson Assembly? How does it work? Takes how long? Is it directional?
it is when have insert and vector that have overlapping sequences. Alternatively, you can join many fragements together based on the overlaps, so can join 3 inserts to eachother while adding them into vector. Works by having 5’ exonuclease (creates 3’ overhangs). Then overlap on the overhangs is used to anneal insert to vector, then DNA pol fills and DNA ligase seals nicks. Takes about an hour. It is directional
