Explain, in detail, the amplification and cloning process for expression of vector - 1. Purify the plasmid and amplify your WT gene with PCR
2. Perform a site-directed mutagenesis
a. pair mutagenic primers with WT primers
b. Purify products from PCR, and perform a
second PCR with the WT p...
Explain, in detail, the amplification and cloning process for expression of vector - ✔✔1. Purify the
plasmid and amplify your WT gene with PCR
2. Perform a site-directed mutagenesis
a. pair mutagenic primers with WT primers
b. Purify products from PCR, and perform a
second PCR with the WT primers
3. Clone amplified WT and mutant gene of interest into an expression vector
a. cut product with correct restriction
enzymes
b. Purify
c. Ligate expression vector with WT and
mutant genes
d. transform an E.coli cell with vector through
heat shock
4. check for successful transformation using PCR
5. Express your fusion proteins
Name the role of each chemical during plasmid purification
EDTA:
SDS/NaOH:
KOAc: - ✔✔EDTA: chelates divalent cations (Mg2+)
, SDS: breaks the membrane open
NaOH: denatures chromosomal DNA
together SDS and NaOH lyse the cells
KOAc: neutralizes the reaction, allowing the plasmid to renature
Name the role of each chemical involved in DNA digestion
NaCl:
Tris-HCl:
Mg2+: - ✔✔NaCl: provides correct ionic strength
Tris-HCl: provides proper pH
Mg2+: acts as enzyme cofactor
How do we prevent our vector from re-ligating?
Why do we implement this step? - ✔✔We CIP-treat our vector which removes the 5' phosphate needed
for DNA ligase to form a phosphodiester bond
We add this step because we do not want our vector to ligate without our insert! Our vector will grow on
ampicillin regardless of if our insert is present, so just because we see growth does not confirm our
insert is present
Why is ATP added to the ligation reaction? - ✔✔ATP is a cofactor needed by DNA ligase
What are the six components needed for PCR? - ✔✔1. template DNA
2. sequence-specific primers
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