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Amplification: Linear
DPN1 Digest: Required
Ligation: Not Required
NEB Exponential
Primers: Back-to-back, mutation at center of one
Phosphorylation: Required
Amplification: Exponential
DPN1 Digest: Not Required
Ligation: Required
Sub-cloning
Sub cloning entails the transfer of a gene from cDNA to an expression vector, or the
transfer of genes between expression vectors.
Multiple types of sub cloning are utilized, but the most common type makes use of
restriction enzymes for the highly-specific splicing of DNA fragments
Restriction Enzymes
Utilizes common Restriction sites (i.e. NdeI, NcoI, HindIII, BamHI, NotI,
XhoI, EcoRI) and corresponding enzymes to cut and paste DNA fragments.
Overlap extension PCR can be used to amplify DNA while also adding on
restriction sites. Additional bases should be added before site to maximize efficiency, as
shown here: https://www.neb.com/tools-and-resources/usage-guidelines/cleavageclose-to-the-end-of-dna-fragments.
Advantage: Widely used, diverse options. Disadvantage: Complicated,
may use low efficiency enzymes
Gateway
Gene is transferred from a vector or cDNA into a donor Gateway vectors
using restriction enzymes which can easily transfer the gene to recipient Gateway
vectors.
Advantage: Simplicity. Disadvantage: Lack of diversity in options
TOPO/TA
Gene is amplified using Taq-driven PCR, which leaves an adenosine
nucleotide at the 3 end. TOPO vectors are sold ready-to-ligate with a 5 T overhang and
a 3-fused DNA topoisomerase
Advantage: Very quick and easy. Disadvantage: Cost, Lack of diversity,
Vectors can only be ordered
Isothermal Assembly Reaction (Gibson Assembly)
Uses a 5 exonuclease to digest 5 ends of any dsDNA. Homologous
regions will auto anneal. A polymerase will fill in ssDNA regions and a ligase will fix any
nicks.
Advantage: Very diverse in application as it does not require specific
sequences. Disadvantage:
Type IIS Assembly (Golden Gate/MoClo)
Ligation independent cloning (LIC)
Yeast Mediated/Oligonucleotide Stitching
Vector Development
Origins of Replication
Antibiotic Resistance
MCS cassettes
Phosphorylation
DNA Polymerases
Restriction
Dephosphorylation
Ligation
Purification
Gel
Silica
Transformation
Cell Choice