L16 Alternative Cloning Methods Flashcards
What is the main feature of seamless cloning methods like Gibson Assembly?
they do not leave cloning scars on the recombinant vector
What temperature is Gibson assembly typically performed at?
50℃
What are the three main types of enzyme used in Gibson Assembly?
5’ exonuclease, DNA polymerase, and DNA ligase
What role does the 5’ exonuclease play in Gibson assembly?
it chews ack 5’ ends to expose complementary 3’ overhangs
Why is Gibson assembly considered a seamless technique?
it leaves no scars and maintains the open reading frame
Why is Gibson assembly directional?
because of the design of specific overlapping regions
How many fragments can be assembled in one Gibson assembly reaction?
multiple fragments
What is one disadvantage of Gibson assembly?
it can be hard to troubleshoot as all steps occur in a single tube
What needs to be carefully designed for Gibson assembly to work?
primers with overlapping sequences
What makes Ligation Independent Cloning (LIC) possible?
the 3’–>5’ exonuclease activity of T4 DNA polymerase
What must be included in the PCR primers for LIC?
LIC extensions with complementary overhangs
What component limits T4 DNA polymerase’s exonuclease activity in LIC?
a single dNTP in the reaction mix
In LIC, where does the final ligation of vector and insert occur?
inside the E.coli cell
Why is LIC considered “scarless”?
it creates no additional sequences at the junction site
What is a key advantage of LIC over restriction enzyme cloning?
no need for ligase or restriction enzyme sites
What is one limitation of LIC?
it doesn’t easily allow the addition of tags or fusions
What kind of inserts does LIC work best with?
PCR-amplified inserts
What is a major benefit of LIC in terms of cost?
it is low cost compared to other seamless methods
How is infusion cloning similar to LIC?
both use homologous recombination and exonuclease activity
What type of extension is required on primers in infusion cloning?
15bp sequences homologous to vector ends
What enzyme creates single-stranded ends in Infusion cloning?
the infusion enzyme mix
What is a key benefit of inclusion cloning in terms of speed?
it works in as little as 15 minutes
Why is infusion cloning considered flexible?
it can be used with any vector
What is a major drawback of inclusion cloning?
occasional mutations in the plasmid
