Problem 4: Briefly, explain the steps of the Alkaline Lysis “miniprep”.
Problem 5: Briefly, explain how qPCR can be used to quantify the level of gene expression
Solution
Problem 4 )
Alkaline lysis is a method used in molecular biology , to isolate plasmid DNA or other cell
components such as proteins by breaking the cells open. Bacteria containing the plasmid of
interest is first grown, and then allowed to lyse with an alkaline lysis buffer consisting of a
detergent sodium dodecyl sulfate (SDS) and a strong base sodium hydroxide. The detergent
cleaves the phospholipid bilayer of membrane and the alkali denatures the proteins which are
involved in maintaining the structure of the cell membrane. Through a series of steps involving
agitation, precipitation, centrifugation, and the removal of supernatant, cellular debris is removed
and the plasmid is isolated and purified.
Problem 5 )
PCR
is used directly to amplify genes. It is mainly used to detect alleles I.e variants of a gene that can
be used to
e.g. identify individuals (e.g. DNA fingerprinting)
identify subpopulations (e.g. tracking ivory poaching)
determine which variants are associated with a condition (e.g. drug efficacy)y
real-time polymerase chain reaction (RT-PCR), also known as quantitative polymerase chain
reaction (qPCR), is a laboratory technique of molecular biology based on the polymerase chain
reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR, i.e. in
real-time, and not at its end, as in conventional PCR. Real-time PCR can be used quantitatively
(quantitative real-time PCR), and semi-quantitatively, i.e. above/below a certain amount of DNA
molecules (semi quantitative real-time PCR).
Two common methods for the detection of PCR products in real-time PCR are: (1) non-specific
fluorescent dyes that intercalate with any double-stranded DNA, and (2) sequence-specific DNA
probes consisting of oligonucleotides that are labelled with a fluorescent reporter which permits
detection only after hybridization of the probe with its complementary sequence..
Problem 4 Briefly, explain the steps of the Alkaline Lysis “minipre.pdf
1. Problem 4: Briefly, explain the steps of the Alkaline Lysis “miniprep”.
Problem 5: Briefly, explain how qPCR can be used to quantify the level of gene expression
Solution
Problem 4 )
Alkaline lysis is a method used in molecular biology , to isolate plasmid DNA or other cell
components such as proteins by breaking the cells open. Bacteria containing the plasmid of
interest is first grown, and then allowed to lyse with an alkaline lysis buffer consisting of a
detergent sodium dodecyl sulfate (SDS) and a strong base sodium hydroxide. The detergent
cleaves the phospholipid bilayer of membrane and the alkali denatures the proteins which are
involved in maintaining the structure of the cell membrane. Through a series of steps involving
agitation, precipitation, centrifugation, and the removal of supernatant, cellular debris is removed
and the plasmid is isolated and purified.
Problem 5 )
PCR
is used directly to amplify genes. It is mainly used to detect alleles I.e variants of a gene that can
be used to
e.g. identify individuals (e.g. DNA fingerprinting)
identify subpopulations (e.g. tracking ivory poaching)
determine which variants are associated with a condition (e.g. drug efficacy)y
real-time polymerase chain reaction (RT-PCR), also known as quantitative polymerase chain
reaction (qPCR), is a laboratory technique of molecular biology based on the polymerase chain
reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR, i.e. in
real-time, and not at its end, as in conventional PCR. Real-time PCR can be used quantitatively
(quantitative real-time PCR), and semi-quantitatively, i.e. above/below a certain amount of DNA
molecules (semi quantitative real-time PCR).
Two common methods for the detection of PCR products in real-time PCR are: (1) non-specific
fluorescent dyes that intercalate with any double-stranded DNA, and (2) sequence-specific DNA
probes consisting of oligonucleotides that are labelled with a fluorescent reporter which permits
detection only after hybridization of the probe with its complementary sequence.
